Method for treating schizophrenia

ABSTRACT

The invention is directed to a method for treating the 22q11 deletion syndrome (22q11 DS) and schizophrenia (SCZ) by replenishment of decreased levels of miR-338-3p in thalamic neurons.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/740,687, filed Sep. 28, 2017, which is the U.S. National Phaseapplication under 35 U.S.C. § 371 of International Patent ApplicationNo. PCT/US2016/040414, filed on Jun. 30, 2016, and claims priority toU.S. Provisional Application No. 62/186,890, filed on Jun. 30, 2015, allof which applications are incorporated by reference in their entireties.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.MH097742 and MH095810 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 24, 2016, isnamed 243734_000079_SL.txt and is 13,687 bytes in size.

FIELD OF THE INVENTION

The invention is directed to a method for treating the 22q11 deletionsyndrome (22q11 DS) and schizophrenia (SCZ) by replenishment ofdecreased levels of miR-338-3p in thalamic neurons.

BACKGROUND OF THE INVENTION

Auditory hallucinations and other positive symptoms of schizophrenia(SCZ) such as delusions, disorganized thought, and psychosis typicallyappear during late adolescence or early adulthood^(1,2) and arealleviated in most patients by antipsychotics that inhibit D2 dopaminereceptors (DRD2s)³⁻⁵. The mechanisms of such late onset ofschizophrenia's positive symptoms and underlying neuronal circuitsremain unknown. Thalamocortical (TC) projections to the auditory cortex(ACx) emerged as a circuit specifically disrupted in mouse models of22q11 deletion syndrome (22q11DS)^(6,7). This disorder is a leadinggenetic cause of schizophrenia⁸⁻¹⁰ and instigated by the hemizygousdeletion of multiple genes (1.5-3 Mb) of the q (long) arm of chromosome22 in humans¹¹. Deletion of one 22q11DS gene, the microRNA(miRNA)-processing gene Dgcr8, leads to high levels of Drd2 in theauditory thalamus, TC disruption, abnormal sensitivity of TC projectionsto antipsychotics, and deficits in acoustic-startle responsescharacteristic of schizophrenia⁶, through miRNA depletion¹²⁻¹⁴.

Several studies have indicated that drug-naïve schizophrenic patientshave elevated levels of DRD2s in their brains^(25,26). Drd2 upregulationleads to deficits in TC synaptic transmission and acoustic startle andalso renders TC projections sensitive to antipsychotics. Antipsychoticsthat effectively treat only positive symptoms but not cognitive ornegative symptoms of the disease²⁷, eliminate synaptic deficits at TCprojections and acoustic-startle deficiency in 22q11DS mice⁶.Antipsychotics alleviate positive symptoms of schizophrenia throughsystemic inhibition of DRD2, which is accompanied by multiple andsometimes devastating side effects, such as blood abnormalities, weightgain, abnormal movements (such as the movements as with Parkinson'sdisease) and many others^(2,28).

The 22q11.2 deletion syndrome (22q11DS) is associated with high risk ofdeveloping schizophrenia symptoms, including psychosis, later in life.Auditory thalamocortical projections recently emerged as a circuitspecifically disrupted in 22q11DS mouse models. Haploinsufficiency ofthe microRNA-processing gene Dgcr8 results in the elevation of thedopamine receptor Drd2 in the auditory thalamus, an abnormal sensitivityof thalamocortical projections to antipsychotics, and an abnormalacoustic-startle response. These auditory abnormalities have a delayedonset in 22q11DS mouse models and are associated with age-dependentreduction of the microRNA miR-338-3p, which targets Drd2 and is enrichedin the thalamus of humans and mice. Replenishing depleted miR-338-3p inthe mature 22q11DS mice rescued the thalamocortical abnormalities, andmiR-338-3p deletion/knockdown mimicked thalamocortical and behavioraldeficits and eliminated their age dependence. Thus, miR-338-3p depletionis necessary and sufficient to disrupt auditory thalamocorticalsignaling in 22q11DS mouse models and may therefore mediate thepathogenic mechanism of 22q11DS-related psychosis and control its lateonset.

The 22q11DS is considered a leading genetic cause of schizophrenia⁴⁶⁻⁴⁸.Schizophrenia develops in 23% to 43% of individuals with 22q11DS⁴⁹⁻⁵⁴,most of whom experience psychosis^(55,56). Furthermore, 30% to 50% ofnonschizophrenic individuals with 22q11DS demonstrate subthresholdsymptoms of psychosis⁵⁷. Nonpsychotic behavioral abnormalities arepresent from early adulthood in 22q11DS^(58, 59), but psychotic symptomsand schizophrenia are delayed until adulthood^(54, 60). It remainsunclear why the onset of psychotic symptoms is so delayed. Inschizophrenic patients, auditory hallucinations and other psychoticsymptoms are similarly delayed until late adolescence or earlyadulthood^(61, 62), are present in 60% to 90% of cases⁶³, and are oftenalleviated by antipsychotics that inhibit D2 dopamine receptors(DRD2s)⁶⁴⁻⁶⁷. Given the germline occurrence of deleted genes in 22q11DS,it is not clear why the onset of positive symptoms is delayed.

Recently, Dgcr8 emerged as a culprit gene responsible for severalneuronal phenotypes observed in mouse models of 22q11DS⁶⁸⁻⁷⁰, includingthe disruption of synaptic transmission at TC projections to the ACx³⁹.Dgcr8 is part of the microprocessor complex that mediates the biogenesisof microRNAs (miRNAs), small RNAs that negatively regulate theexpression of complementary mRNAs and protein translation⁷¹. Dgcr8haploinsufficiency in 22q11DS leads to depletion of miRNAs and theresultant upregulation of respective targets, which in turn disruptssynaptic transmission, synaptic plasticity, and proper functioning ofneural circuits⁷². In adult 22q11DS mouse models, Dgcr8haploinsufficiency is sufficient to upregulate Drd2 mRNA and protein inthe auditory thalamus, which causes auditory abnormalities that includedecreased glutamatergic synaptic transmission at TC projections to theACx and deficient prepulse inhibition (PPI) of the acoustic-startleresponse³⁹. Abnormally high levels of Drd2 in the thalamus of 22q11DSmice increase TC projection sensitivity to Drd2 antagonists, includingantipsychotics. As a consequence, auditory synaptic and behavioralabnormalities of 22q11DS mice are rescued by antipsychotics³⁹.

It was tested whether TC disruption follows the same age-dependenttrajectory as psychosis in patients with 22q11DS or schizophrenia anddetermined the molecular underpinnings of TC disruption in 22q11DS mice.Similar to psychotic symptoms, TC disruption of synaptic transmissionhad a delayed onset. In a series of miRNA and physiological screens, thethalamus-enriched Drd2-targeting miR-338-3p was identified as that whichmediates the Dgcr8-Drd2 mechanism of TC disruption. It is also shownthat miR-338-3p is depleted in mouse models of 22q11DS and schizophrenicpatients and that replenishment of miR-338-3p in the auditory thalamusrescued the TC deficits in 22q11DS mouse models. Lastly, evidence ispresented showing that miR-338-3p is a key controller of the late onsetof TC disruption in 22q11DS mice.

SUMMARY OF THE INVENTION

There is a great need in the art to develop effective treatments forschizophrenia and the 22q11 deletion syndrome, including treatment ofpositive symptoms of schizophrenia, such as, e.g., hallucinations,delusions, disorganized thought, and psychosis. There is also a greatneed to develop diagnostic methods for schizophrenia. The presentinvention addresses these and other needs by providing methods based onmicroRNA miR-338-3p.

In one aspect, the invention provides a method for treatment and/orprevention of schizophrenia in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of (i) miR-338-3p or a mimic or a functional derivative thereof(including functional fragments of miR-338-3p and their derivatives), or(ii) a vector expressing said miR-338-3p or mimic or functionalderivative thereof, or (iii) an agent capable of increasing the level oractivity of miR-338-3p.

In a related aspect, the invention provides a method for treatment of22q11 deletion syndrome in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of (i) miR-338-3p or a mimic or a functional derivative thereof(including functional fragments of miR-338-3p and their derivatives), or(ii) a vector expressing said miR-338-3p or mimic or functionalderivative thereof, or (iii) an agent capable of increasing the level oractivity of miR-338-3p.

In another related aspect, the invention provides a method for treatmentand/or prevention of a positive symptom of schizophrenia (e.g.,hallucinations, delusions, disorganized thought, or psychosis) in asubject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of (i) miR-338-3p or a mimicor a functional derivative thereof (including functional fragments ofmiR-338-3p and their derivatives), or (ii) a vector expressing saidmiR-338-3p or mimic or functional derivative thereof, or (iii) an agentcapable of increasing the level or activity of miR-338-3p.

In one embodiment of any of the above methods, the miR-338-3p or mimicor derivative thereof comprises the sequence UCCAGCAUCAGUGAUUUUGUUG (SEQID NO: 1). In one embodiment of any of the above methods, the miR-338-3por mimic or derivative thereof consists of the sequenceUCCAGCAUCAGUGAUUUUGUUG (SEQ ID NO: 1).

In one embodiment of any of the above methods, the vector is selectedfrom the group consisting of adeno-associated virus (AAV) vectors,lentivirus vectors and Sindbis virus vectors.

In one embodiment of any of the above methods, the expression ofmiR-338-3p or mimic or functional derivative thereof in the vector iscontrolled by a promoter selected from the group consisting of Synapsinpromoter, CMV promoter, β-actin promoter, and CamKIIa promoter.

In one embodiment of any of the above methods, the administration issystemic. In another embodiment of any of the above methods, theadministration is intranasal.

In one embodiment of any of the above methods, the administration istargeted to the thalamus.

In one embodiment of any of the above methods, the administrationresults in an increase in the level of miR-338-3p in thalamic neurons ofthe subject to the level found in healthy subjects.

In one embodiment of any of the above methods, the administrationresults in a decrease in sensitivity of thalamic neurons to anantipsychotic agent (e.g., haloperidol, clozapine, olanzapine, oranother antipsychotic agent inhibiting a D2 dopamine receptor).

In one embodiment of any of the above methods, the subject has adecreased level of miR-338-3p in thalamic neurons as compared to acontrol (e.g., a predetermined standard, or the level of miR-338-3p inthalamic neurons of a healthy age- and gender-matched subject or anaverage value for several such subjects).

In one embodiment of any of the above methods, the method furthercomprises determining the level of miR-338-3p in thalamic neurons or abodily fluid sample obtained from the subject (e.g., blood [e.g., wholeblood, blood lymphocytes, peripheral blood mononuclear cells (PBMCs),blood plasma, or blood serum], urine, saliva, or cerebrospinal fluid[CSF]). In one specific embodiment, the level of miR-338-3p isdetermined using a method selected from the group consisting ofhybridization, array-based assays, PCR-based assays (e.g., qPCR), andsequencing. In one specific embodiment, the level of miR-338-3p isdetermined prior to the administration of the treatment. In one specificembodiment, the level of miR-338-3p is determined both prior and afterthe administration of the treatment.

In one embodiment of any of the above methods, the method furthercomprises administering to the subject an additional treatment agent. Inone specific embodiment, the additional treatment agent is anantipsychotic (e.g., haloperidol, clozapine, olanzapine, or anotherantipsychotic agent inhibiting a D2 dopamine receptor).

In a separate aspect, the invention provides a method for determiningefficacy of a treatment for schizophrenia or 22q11 deletion syndrome ina subject, the method comprising:

(a) determining the level of miR-338-3p in thalamic neurons or a bodilyfluid sample obtained from the subject before the treatment,

(b) determining the level of miR-338-3p in thalamic neurons or a bodilyfluid sample obtained from the subject after the treatment,

(c) comparing the levels determined in steps (a) and (b), and

(d) determining that the treatment is effective if the level ofmiR-338-3p in thalamic neurons or the bodily fluid of the subject hasincreased after the treatment.

In another aspect, the invention provides a method for determining thelikelihood of developing a positive symptom of schizophrenia (e.g.,hallucinations, delusions, disorganized thought, or psychosis) in asubject, the method comprising:

(a) determining the level of miR-338-3p in thalamic neurons or a bodilyfluid sample obtained from the subject,

(b) comparing the level determined in step (a) to a control level, and

(c) determining that the subject is at risk of developing a positivesymptom of schizophrenia if the level of miR-338-3p in thalamic neuronsor the bodily fluid sample obtained from the subject is lower than thecontrol level.

In one embodiment of any of the above diagnostic methods, the control isa predetermined standard, or the level of miR-338-3p in thalamic neuronsof a healthy age- and gender-matched subject or an average value forseveral such subjects.

In one embodiment of any of the above diagnostic methods, the bodilyfluid is selected from the group consisting of blood (e.g., whole blood,blood lymphocytes, peripheral blood mononuclear cells (PBMCs), bloodplasma, or blood serum), urine, saliva, and cerebrospinal fluid (CSF).

In one embodiment of any of the above diagnostic methods, the level ofmiR-338-3p is determined using a method selected from the groupconsisting of hybridization, array-based assays, PCR-based assays (e.g.,qPCR), and sequencing.

In one embodiment of any of the above diagnostic methods, prior todetermining miR-338-3p level, miRNA is purified from the sample isolatedfrom the subject.

In one embodiment of any of the above diagnostic methods, the methodfurther comprises the step of reducing or eliminating degradation ofmiRNA.

In one embodiment of any of the above diagnostic methods, the miR-338-3pcomprises the sequence UCCAGCAUCAGUGAUUUUGUUG (SEQ ID NO: 1). In oneembodiment of any of the above diagnostic methods, the miR-338-3pconsists of the sequence UCCAGCAUCAGUGAUUUUGUUG (SEQ ID NO: 1). In oneembodiment of any of the above methods of the invention, the subject ishuman. In another embodiment of any of the above methods of theinvention, the subject is an experimental animal model.

In a related aspect, the invention provides a kit for determining thelikelihood of developing a positive symptom of schizophrenia comprisingprimers and/or probes specific for miR-338-3p.

In another related aspect, the invention provides a kit for determiningefficacy of a treatment for schizophrenia or 22q11 deletion syndromecomprising primers and/or probes specific for miR-338-3p.

In one embodiment, the kits of the invention comprise miRNA isolationand/or purification means.

In one embodiment, the kits of the invention comprise instructions foruse. In another aspect, the invention provides the use of (i) miR-338-3por a mimic or a functional derivative thereof (including functionalfragments of miR-338-3p and their derivatives), or (ii) a vectorexpressing said miR-338-3p or mimic or functional derivative thereof, or(iii) an agent capable of increasing the level or activity of miR-338-3pin the manufacture of a medicament in the treatment and/or prevention ofschizophrenia.

In yet another aspect, the invention provides the use of (i) miR-338-3por a mimic or a functional derivative thereof (including functionalfragments of miR-338-3p and their derivatives), or (ii) a vectorexpressing said miR-338-3p or mimic or functional derivative thereof, or(iii) an agent capable of increasing the level or activity of miR-338-3pin the manufacture of a medicament in the treatment of 22q11 deletionsyndrome.

In a further aspect, the invention provides the use of (i) miR-338-3p ora mimic or a functional derivative thereof (including functionalfragments of miR-338-3p and their derivatives), or (ii) a vectorexpressing said miR-338-3p or mimic or functional derivative thereof, or(iii) an agent capable of increasing the level or activity of miR-338-3pin the manufacture of a medicament in the treatment and/or prevention ofa positive symptom of schizophrenia.

In one embodiment of any of the above aspects, the invention furtherprovides the use of In another aspect, the invention provides apharmaceutical composition comprising miR-338-3p or a mimic or afunctional derivative thereof (including functional fragments ofmiR-338-3p and their derivatives) and a pharmaceutically acceptablecarrier or excipient. In one embodiment, said composition is suitablefor intranasal administration. In another embodiment, said compositionis suitable for systemic administration.

In a related aspect, the invention provides a pharmaceutical dosage formcomprising miR-338-3p or a mimic or a functional derivative thereof(including functional fragments of miR-338-3p and their derivatives) anda pharmaceutically acceptable carrier or excipient. In one embodiment,said dosage form is suitable for intranasal administration. In anotherembodiment, said dosage form is suitable for systemic administration.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in the following description, claimsand drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a -1 n. Adult onset of antipsychotics sensitivity and synaptictransmission disruption in auditory TC projections of mouse models of22q11DS. (a) Map of 22q11DS orthologs deleted in Df(16)1/+ mice. (b)Illustration of voltage-clamp recordings of thalamorecipient L3/4pyramidal neurons in TC slices. TC projections are shown in red. ACx,auditory cortex; TC, thalamocortical; MGv, ventral part of the medialgeniculate nuclei. (c, d, e) Late onset of Drd2 elevation in theauditory thalamus and TC synaptic transmission disruption in 22q11DSmice. (c, d) Input-output relations between stimulation intensity andEPSCs at TC projections in the ACx of 2- (c) or 4-month-old (d) WT(black) mice and Df(16)1/+ (white) mice. (e) Drd2 transcript levels inthe MGV of 2- and 4-month-old WT (left black bar) and Df(16)1/+ mice(right gray bar) (numbers of mice are shown inside columns). (f, g) Theeffect of haloperidol on TC EPSCs in 2- (f) and 4-month-old (g) WT(black) and Df(16)1/+ (white) littermates. Haloperidol-inducedpercentage change (ΔH) in the slope of TC EPSCs relative to baseline(before haloperidol application; dashed line). (h) The ΔH as a functionof mouse age in WT (black) and Df(16)1/+ (white) littermates. (i, j) Theeffect of haloperidol on TC EPSCs in 2- (i) and 4-month-old (j) WT(black) and Dgcr8^(+/−) (gray) littermates. (k) The ΔH as a function ofmouse age in WT (black) and Dgcr8^(+/−) (gray) littermates. (1) AverageDrd2 mRNA levels normalized to Gapdh in the auditory thalamus of 2- and4-month-old WT (left black bar) and Dgcr8^(+/−) littermates (right graybar). (m, n) Mean PPI of maximal acoustic-startle response in 2- (m) and4-month-old (n) WT (left black bar) and Dgcr8^(+/−) (right gray bar)littermates. For the 2-month experiment (m), 23 WT mice and 22Dgcr8^(+/−) mice were used; in the 4-month experiment (n), 36 WT miceand Dgcr8^(+/−) mice were used. Scale bars, 50 pA, 10 ms. Insets showrepresentative EPSCs before (1) and after (2) haloperidol application.Numbers of mice or neurons are shown inside the columns or parenthesis,with the number of WT mice/neurons first. Data are represented as themean±SEM. SPL (sound pressure level), *p<0.05.

FIG. 2a -2 j. Identification of Drd2-targeting miR-338-3p in theauditory thalamus. (a-d) Volcano plots of miRNA microarray data from theauditory thalamus of 2- (a, c) and 4-month-old (b, d) WT and Df(16)1/+(a, b) and WT and Dgcr8^(+/−) (c, d) male littermates. The differencebetween miRNA levels in WT and mutants was considered significant ifp<0.01 and loge fold change (FC)>±0.2. Symbol size represents the miRNAexpression level in the microarray. Note miR-338-3p had the highestexpression among all predicted Drd2-targeting miRNAs. (e) Diagram of themouse Drd2 3′UTR (XM_006509996.2; SEQ ID NO: 43) with seed sites for the5 miRNAs indicated. (f) Experimental design of a recombinant AAVencoding a chimeric construct overexpressing an miRNA of interest (top)injected into the mouse MGv (bottom). (g) GFP expressed specifically inthe auditory TC projections after in vivo injection of recombinant AAV.(h) Haloperidol sensitivity of TC projections in 4-month-old WT mice(left black bar) and Df(16)1/+ mice (right gray bar) previously injectedwith AAVs encoding different miRNAs or GFP. Only miR-338-3p eliminateshaloperidol sensitivity in TC projections of Df(16)1/+ mice. Numbers ofmice are indicated in parentheses above each bar; the left-hand numberis the number of WT mice used and the right-hand number is the number ofDf(16)1/+ mice used. (i) Relative average levels of miRNA expression inthe thalamus, hippocampus, and cortex of WT mice (normalized to theaverage of three housekeeping genes: U6, snoRNA202, and snoRNA234). OnlymiR-338-3p shows enrichment in the thalamus. (j) Mean relativemiR-338-3p levels (normalized to U6) in the postmortem MGv and ACxtissues from patients with schizophrenia (SCZ) (right gray bar) andhealthy controls (left black bar). Six healthy controls and 6 patientswith SCZ were used.

FIG. 3a -3 d. Replenishment of miR-338-3p in the auditory thalamusrescues deficits in synaptic transmission and presynapticneurotransmitter release at TC projections of 22q11DS mouse models. (a)In vivo infection of MGv relay neurons with AAV-GFP-miR-338-3p orAAV-GFP. (b) GFP expression (gray) in cell bodies in the MGv (left) andin projections to the thalamorecipient L3/4 layer of the ACx (right). Apatch pipette and part of an L3/4 pyramidal neuron filled with Alexa 594are shown in gray. (c, d) Input-output relations between stimulationintensity and EPSCs (c) and PPR (d) at TC projections in the ACx of 4-to 5-month-old WT and Df(16)1/+ mice injected with eitherAAV-GFP-miR-338-3p or AAV-GFP. WT-GFP=black; WT-miR-338-3p=darkest grayin panel c and lightest gray in panel d; Df(16)1/+−GFP=medium gray;Df(16)1/+− miR-338-3p=gray. Insets show representative EPSCs. Scale bar,20 ms, 50 pA. Numbers of neurons are shown in parentheses, with thenumber of WT neurons first. Data are represented as the mean±SEM.*p<0.001.

FIG. 4a -4 p. The depletion of knockout of miR-338 replicates the TCdeficiency of Df(16)1/+ mice. (a) AAV expressing a miR-338-3p spongeconstruct with multiple binding sites to miR-338-3p in the GFP 3′UTR,under control of the CamKIIα promoter. Sequences for the miR-338-3psponge and scrambled control are shown below. Bold text indicatesseed-site sequence. (b) Relative Drd2 mRNA levels after infection of MGvexcitatory neurons in WT mice with an AAV encoding a scrambled control(left black bar) or miR-338-3p sponge (right gray bar). (c) Normalizedmean TC EPSCs before and after application of haloperidol in WT miceafter infection of MGv neurons with AAVs encoding a scrambled control(black)or miR-338-3p sponge (light gray). Insets show representativeEPSCs. (d) Generation of miR-338 KO mice. (e) Normalized levels ofmiR-338-3p and Drd2 in the auditory thalamus of WT (left black bar),miR-338^(+/−) (middle dark gray bar), and miR-338^(−/−) (right lightgray bar) mice. (f) Simultaneous recordings of EPSCs in L3/4 pyramidalneurons evoked by electrical stimulation of the thalamocortical (TC) andcorticocortical (CC) projections. (g, h) Input-output relations betweenelectrical stimulation intensity and EPSCs at TC projections (g) and CCprojections (h) in the ACx of 4-month-old WT (black) and miR-338^(+/−)(white) mice. (i-l) PPR (i, j) and NMDAR/AMPAR ratio (k, l) ofelectrically evoked EPSCs measured at TC (i, k) and CC projections (j,l) of 4-month-old WT (black) and miR-338^(+/−) (gray or white) mice. WTmice (n=26) are represented by the bottom black line and miR-338^(+/−)mice (n=22) are represented by the upper gray line in i. WT mice arerepresented by the left black bar and miR-338^(+/−) mice are representedby the right gray bar in k, l, with numbers of neurons indicated in thebars. (m) Optogenetic experiments in TC slices. ChR2 was expressed inthe MGv, under control of the CamKIIα promoter. (n-p) Input-outputrelations (n), PPR (o), and NMDAR/AMPAR ratio (p) of optically evokedEPSCs (oEPSC) measured at TC projections of 4-month-old WT (black) andmiR-338^(+/−) (white) mice. WT mice are represented by the left blackbar and miR-338^(+/−) mice are represented by the right gray bar in p,with numbers of neurons indicated in the bars. Insets showrepresentative AMPAR-mediated (−70 mV holding membrane potential) andNMDAR-mediated (+40 mV holding membrane potential) EPSC and oEPSCtraces. Scale bars (if not noted otherwise), 20 ms, 50 pA. Numbers ofneurons are shown inside the columns or parentheses, with the number ofWT neurons first. Data are represented as the mean±SEM. *p<0.05.

FIG. 5a -5 f. Probability of glutamate release is reduced at TCprojections of miR-338^(+/−) mice. (a) An L3/4 pyramidal neuron filledwith Fluo-5F and Alexa 594 through a patch pipette (left) to visualizesynaptically evoked calcium transients inside dendritic spines (right).Gray line represents the line scan. (b) Calcium transients in adendritic spine in response to a single thalamic stimulation (arrows)repeated 10 times at 0.1 Hz. (c) Location of active TC inputs ondendritic trees of L3/4 pyramidal neurons spines. (0;0), somacoordinates (apical dendrites pointing upwards); black circles representdata from WT mice while white circles represent data from miR-338^(+/−)mice. (d-f) Average distances from soma to active TC inputs (d), calciumtransient peak amplitudes (e), and probabilities (f) in response to 10to 20 single TC stimulations. WT mice are represented by the left blackbar and miR-338^(+/−) mice are represented by the right gray bar in d-f.Numbers of spines are shown in parentheses (WT=27 spines,miR-338^(+/−)=32 spines). Data are represented as the mean±SEM. *p<0.01.

FIG. 6a -6 g. Deletion of miR-338 in mice eliminates age dependency forantipsychotics sensitivity and replicates 22q11DS phenotypes. (a)Average TC EPSCs before (1) and during (2-3) application of theDrd2-specific inhibitor L-741,626 and haloperidol in 4-month-old WT(black) and miR-338^(+/−) (white) mice. (b) Haloperidol sensitivity (ΔH)in WT (black) and miR-338^(+/−) (white) mice between 1.5 and 4 months ofage. (c, d) Mean TC EPSCs before (1) and after (2) haloperidol in2-month-old WT (black) and miR-338^(+/−) (white) mice that receivedcontrol (c) or Drd2 siRNA injected into their MGv (d). Insets showrepresentative EPSCs. (e-g) Mean PPI of maximal acoustic startleresponse in 1.5- (e), 2- (f), and 4-month-old (g) WT and miR-338^(+/−)littermates. WT mice are represented by the left black bar andmiR-338^(+/−) mice are represented by the right gray bar in e-g. Numbersof mice are shown in parentheses (e, WT=22, miR-338^(+/−)=21; f, WT=22,miR-338^(+/−)=21; g, WT=21, miR-338^(+/−)=20). Numbers of neurons ormice are shown inside parentheses, with the number of WT mice/neuronsfirst. Data are represented as the mean±SEM. SPL, sound pressure level.*p<0.05.

FIG. 7. QRT-PCR verification of depletion of five Drd2-targeting miRNAsin the MGv of Dgcr8^(+/−) mice. Relative expression levels of the U6loading control and Drd2-targeting miRNAs in the auditory thalamus of 2-and 4-month-old WT and Dgcr8^(+/−) littermates. The left-most (lightestgray) bar for each miRNA corresponds to 2-month WT mice, followed by alighter gray bar corresponding to 2-month Dgcr8^(+/−) littermates,followed by a black bar corresponding to 4-month WT mice, followed by adark gray bar corresponding to 4-month Dgcr8^(+/−) littermates. Data arenormalized to the 2-month-old WT levels for each miRNA. Data arerepresented as the mean±SEM. *p<0.05.

FIG. 8a -8 k. MGv restoration of miR-338-3p but not other Drd2-targetingmiRNAs eliminates abnormal haloperidol sensitivity of TC projections in22q11DS mice. (a-e) Mean levels of miR-337-5p (a), miR-338-3p (b),miR-335-5p (c), miR-337-3p (d), and miR-335-3p (e) were normalized tothe U6 loading control in the MGv of 4-month-old WT and Df(16)1/+ miceinjected with AAVs overexpressing a control vector (GFP) or respectivemiRNAs (n=3-7 mice per experiment). The left black bar corresponds to WTmice while the right gray bar corresponds to Df(16)1/+ mice in each ofa-e. (f-k) Mean TC EPSCs were normalized to baseline before and afterhaloperidol in 4-month-old WT and Df(16)1/+ mice injected with AAVsoverexpressing a control vector (GFP) (f), miR-337-5p (g), miR-338-3p(h), miR-335-5p (i), miR-337-3p (j), or miR-335-3p (k) into the MGv. Theblack circles correspond to WT mice while the dark gray circlescorrespond to Df(16)1/+ mice in each of f-k. Insets show representativetraces before (1) and during (2) the application of haloperidol.*p<0.05.

FIG. 9. Validation of the miR-338-3p sponge. Luminescence activitymeasured with the luciferase reporter vector alone, cloned into theluciferase reporter vector miRNA sponges containing 4 to 26 seed sites,or scrambled sites in the presence of control pcDNA (right dark graybar), miR-338-3p (left black bar), or miR-185-5p (middle light graybar). Based on these experiments, the miR-338-3p sponge with 12 seedsites was chosen for in vivo experiments.

FIG. 10a -10 e. The miR-338-targeted deletion in mice does not affectthe expression of the miR-338 host gene Aatk or mouse development. (a,b) Normalized average levels of Aatk mRNA in the MGv of 2- (light graybar) and 4-month-old (black bar) WT mice (a) and in 4-month-old WT (leftblack bar), miR-338^(+/−) (middle dark gray bar), and miR-338^(−/−)(right light gray bar) littermates (b). Numbers of mice are shown insidethe columns. (c) Relative levels of miR-338-3p, miR-338-5p, miR-3065-3p,and miR-3065-5p in the MGv of WT (left black bar), miR-338^(+/−) (middledark gray bar), and miR-338^(−/−) (right light gray bar) littermates(3-4 mice/genotype). (d, e) Representative image (d) and average bodyweight (e) of WT (left black bar), miR-338^(+/−) (middle dark gray bar),and miR-338^(−/−) (right light gray bar) male littermates (1.5-3 monthsof age). Numbers of mice are shown inside the columns. *p<0.05.

FIG. 11a -11 c. Deletion of miR-338 eliminates age dependency for TCsensitivity to antipsychotics. (a-c) Normalized mean TC EPSCs before (1)and after (2) haloperidol application were measured in the MGvs of WT(black) or miR-338^(+/−) (white) mice at 4 months (a), 2 months (b), or1.5 months (c). Numbers of neurons are shown in parentheses. Insets showrepresentative TC EPSC traces before (1) and after (2) haloperidolapplication. *p<0.05.

FIG. 12a -12 b. Abnormal sensitivity of TC projections in miR-338+/−mice to the antipsychotics clozapine and olanzapine. (a,b) Normalizedmean TC EPSCs before (1) and after (2) 40 μM clozapine (a) and 1 μMolanzapine (b) in 3.5-month old WT (black) and miR-338^(+/−) (white)mice. Numbers of neurons are shown in parenthesis, with the number of WTneurons first. Insets show representative TC EPSC traces before andafter haloperidol application. *P<0.05.

FIG. 13a -13 b. Local depletion of miR-338-3p in the MGv renders TCprojections to the ACx sensitive to antipsychotics at a younger age. (a)In vivo expression of the miR-338-3p sponge (light gray) or scrambledcontrol (black) after injection into the MGv. (b) Normalized mean TCEPSCs before (1) and after (2) application of haloperidol in 2-month-oldWT mice. Numbers of neurons are shown in the parentheses, with thenumber of WT neurons first. Insets show representative TC EPSC tracesbefore (1) and after (2) haloperidol application. *p<0.05.

FIG. 14a -14 c. Normal hearing in miR-338^(+/−) mice. (a-c) Auditorybrainstem responses in WT (black) and miR-338^(+/−) (white) mice at 1.5months (a), 2 months (b), and 4 months (c). Numbers of mice are shown inparentheses, with the number of WT mice first.

FIG. 15 illustrates that an overexpression of miR-338-3p reduces Drd2levels in the MGv. Average levels of Drd2 mRNA normalized to U6 in theMGv of 4-month-old WT (left black bar) and Df(16)1/+ (right gray bar)mice injected with AAVs overexpressing a control vector (GFP) ormiR-338-3p. *P<0.05.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on an unexpected discovery by theinventor that miR-338-3p miRNA is depleted in 22q11 deletion syndrome(which in 30% cases leads to schizophrenia) and this leads to abnormalelevation of Drd2 in the thalamus. This renders thalamic neuronssensitive to antipsychotics.

As further demonstrated herein, replenishment of miR-338-3p normalizesthe Drd2 level and rescues abnormal function of thalamic neurons andtheir abnormal sensitivity to antipsychotics. Thalamic knockdown ordeletion of miR-338-3p mimics 22q11DS molecular, synaptic, andbehavioral auditory abnormalities, and more importantly, does soregardless of age. This result suggests that thalamic miR-338-3p is thecrucial mediator and the late-onset controller of a pathogenic pathwayunderlying the positive symptoms of schizophrenia.

Based on these observations, the invention provides a targeted therapyagainst positive symptoms of schizophrenia that is devoid ofside-effects attributable to antipsychotics, which therapy comprisesreplenishment of miR-338-3p in the thalamus.

Definitions

As used herein, the term “schizophrenia” includes a condition generallydescribed as schizophrenia or a condition having symptoms relatedthereto. Schizophrenia can be considered a disease with a spectrum ofmanifestations with various threshold levels. Symptoms of schizophreniamay appear in a range of related disorders including classicalschizophrenia as well as dementia, bipolar disorder, obsessivecompulsive disorder (OCD), panic disorder, phobias, acute stressdisorder, adjustment disorder, agoraphobia without history of panicdisorder, alcohol dependence (alcoholism), amphetamine dependence, briefpsychotic disorder, cannabis dependence, cocaine dependence, cyclothymicdisorder, delirium, delusional disorder, dysthymic disorder, generalizedanxiety disorder, hallucinogen dependence, major depressive disorder,nicotine dependence, opioid dependence, paranoid personality disorder,Parkinson's disease, schizoaffective disorder, schizoid personalitydisorder, schizophreniform disorder, schizotypal personality disorder,sedative dependence, shared psychotic disorder, smoking dependence andsocial phobia.

In the present application, the terms “microRNA”, “miRNA” and “miR” areused interchangeably to refer to a class of small approximately 20-25 ntlong non-coding RNA molecules. They play important roles in theregulation of target genes through sequence-specific hybridization tothe 3′ untranslated region (UTR) of messenger RNAs (mRNA) to represstheir translation or regulate degradation (Griffiths Jones Nucleic AcidsResearch, 2006, 34, Database issue: D140-D144; Baek et al., Nature455(7209):64 (2008); Selbach et al., Nature 455(7209):58 (2008); Ambros,2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; Cullen,2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5,522-531; and Ying et al., 2004, Gene, 342, 25-28). Most miRNAs aretranscribed by RNA polymerase II from intergenic, intronic orpolycistronic loci to long primary transcripts, called pri-miRNAs.vPri-miRNAs are processed sequentially first in the nucleus (usually bythe Drosha-DGCR8 complex) to approximately 70-100 nt pre-miRNA hairpinstructures and then in the cytoplasm by the Dicer (ribonuclease III-likenuclease enzyme)-TRBP complex to approximately 2-25 nt miRNA duplexes(van Rooij and Kauppinen, EMBO Mol Med., 2014, 6(7): 851-864). In thecytoplasm, miRNA duplexes are incorporated into an Argonauteprotein-containing miRNA-induced silencing complex (miRISC), followed byunwinding of the duplex and retention of the mature miRNA strand inmiRISC, while the complementary strand is released and degraded (vanRooij and Kauppinen, EMBO Mol Med., 2014, 6(7): 851-864). miRNAs guidethe miRISC to target mRNAs by base pairing imperfectly with their3′-UTRs, leading to translational repression and/or degradation of themRNA targets. The miRNA target sites, located in the 3′UTR of mRNAs, areoften imperfectly matched to the miRNA sequence. Frequently, one miRNAcan target multiple mRNAs and one mRNA can be regulated by multiplemiRNAs targeting different regions of the 3′ UTR. The 5′ region ofmiRNA, also known as the “seed” region (nt 2-7), is the most criticalsequence for targeting and function. Unless otherwise noted, the name ofa specific miRNA refers to a mature miRNA sequence. Under currentnomenclature rules, human miRNAs are preceded with the prefix “hsa-”(i.e., an abbreviation for Homo sapiens). Throughout the specificationand figures the hsa- prefix may be dropped for purposes of abbreviation,thus, for example, “hsa-miR-338-3p” and “miR-338-3p” would represent thesame RNA sequence.

The present invention relates to miR-338, which is encoded within theintronic region of the gene for apoptosis-associated tyrosine kinase(AATK). It has been reported that miR-338 may downregulate genes whichhave a downstream negative effect on AATK expression. The sequence ofthe mature human miR-338-3p is 5′UCCAGCAUCAGUGAUUUUGUUG3′ (SEQ ID NO:1).

As defined herein, the term “functional derivative” of a miRNA refers toa miRNA that has less than 100% identity to a corresponding wild-typemiRNA and possesses one or more biological activities of thecorresponding wild-type miRNA. Examples of such biological activitiesinclude, but are not limited to, inhibition of expression of a targetRNA molecule (e g, inhibiting translation of a target mRNA moleculeand/or modulating the stability of a target mRNA molecule) andinhibition of a cellular process associated therewith. These functionalderivatives include species variants and variants that are theconsequence of one or more mutations (e.g., a substitution, a deletion,an insertion) in a miRNA-encoding gene. In certain embodiments, thevariant is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%identical to a corresponding wild-type miRNA. Functional derivativesalso encompass “functional fragments” of miRNA, i.e., portions of miRNAwhich are less than the full-length molecule (and their species andmutant variants) and that possess one or more biological activities of acorresponding wild-type miRNA. In certain embodiments, thebiologically-active fragment is at least about 5, 7, 10, 12, 15, or 17nucleotides in length.

As used herein, the term “miRNA mimic” refers to a double-strandedmiRNA-like RNA fragment. Such miRNA mimic is designed to have its 5′-endbearing a partially complementary motif to the selected sequence in the3′UTR unique to the target mRNA. Once introduced into cells, miRNAmimic, mimicking an endogenous miRNA, can bind to its target mRNA andinhibt its translation and/or modulate its stability. Unlike endogenousmiRNAs, miR-mimics can be made to act in a gene-specific fashion byincreasing the region of perfect complementarity with mRNA 3′ UTR.Often, miRNA mimics are made to harbor chemical modifications to improvestability and/or cellular uptake (Rooij and Kauppinen, EMBO Mol Med.,2014, 6(7): 851-864). In such double-stranded miRNA mimics, the strandidentical to the miRNA of interest is the guide (antisense) strand,while the opposite (passenger or sense) strand is less stable and can belinked to a molecule, such as, e.g., cholesterol, to enhance cellularuptake. In addition, the passenger strand may contain chemicalmodifications to prevent RISC loading, while it is further leftunmodified to ensure rapid degradation. Since the miRISC needs torecognize the guide strand as a miRNA, the chemical modifications thatcan be used for the guide strand are limited. For example, the 2′-fluoro(2′-F) modification helps to protect against exonucleases, hence makingthe guide strand more stable, while it does not interfere with RISCloading (Rooij and Kauppinen, EMBO Mol Med., 2014, 6(7): 851-864).

The terms “vector”, “expression vector”, and “expression construct” areused interchangeably to refer to a composition of matter which can beused to deliver a nucleic acid of interest to the interior of a cell andmediate its expression within the cell. Most commonly used examples ofvectors are autonomously replicating plasmids and viruses (such as,e.g., adenoviral vectors, adeno-associated virus vectors (AAV),lentiviral vectors, Sindbis virus vectors, etc.). An expressionconstruct can be replicated in a living cell, or it can be madesynthetically. In one embodiment, an expression vector comprises apromoter operably linked to a polynucleotide (e.g., a polynucleotideencoding miR-338-3p or its derivative or mimic) which promoter controlsthe initiation of transcription by RNA polymerase and expression of thepolynucleotide. Typical promoters for mammalian cell expression include,e.g., SV40 early promoter, CMV immediate early promoter (see, e.g., U.S.Pat. Nos. 5,168,062 and 5,385,839), mouse mammary tumor virus LTRpromoter, adenovirus major late promoter (Ad MLP), herpes simplex viruspromoter, murine metallothionein gene promoter, and U6 or H1 RNA pol IIIpromoter. Non-limiting examples of promoters useful for expressionmiRNA338-3p in the methods of the present invention include, e.g.,Synapsin promoter (neuron specific), CamKIIa promoter (specific forexcitatory neurons), CMV promoter, and β-actin promoter. These and otherpromoters can be obtained from commercially available plasmids, usingtechniques well known in the art. See, e.g., Sambrook et al., supra.Enhancer elements may be used in association with promoters to increaseexpression levels of the vectors. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence.

Typically, transcription terminator/polyadenylation signals will also bepresent in the expression vector. Examples of such sequences include,but are not limited to, those derived from SV40, as described inSambrook et al., supra, as well as a bovine growth hormone terminatorsequence (see, e.g., U.S. Pat. No. 5,122,458). Additionally, 5′-UTRsequences can be placed adjacent to the coding sequence in order toenhance expression of the same. Such sequences include UTRs whichinclude, e.g., an Internal Ribosome Entry Site (IRES) present in theleader sequences of picornaviruses such as the encephalomyocarditisvirus (EMCV) UTR (Jang et al. J. Virol. (1989) 63:1651-1660. Otheruseful picornavirus UTR sequences include, e.g., the polio leadersequence, hepatitis A virus leader and the hepatitis C IRES.

In certain embodiments of the invention, the cells containing nucleicacid constructs of the present invention may be identified in vitro orin vivo by including a marker in the expression vector. Such markerswould confer an identifiable change to the cell permitting easyidentification of cells containing the expression vector. Usually theinclusion of a drug selection marker aids in cloning and in theselection of transformants, for example, genes that confer resistance toneomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol areuseful selectable markers. Alternatively, enzymes such as herpes simplexvirus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)may be employed. Fluorescent markers (e.g., green fluorescent protein(GFP), EGFP, or Dronpa), or immunologic markers can also be employed.Further examples of selectable markers are well known to one of skill inthe art.

In the context of the present invention insofar as it relates to any ofthe disease conditions recited herein, the terms “treat”, “treatment”,and the like mean to relieve or alleviate at least one symptomassociated with such condition, or to slow or reverse the progression ofsuch condition, or to arrest, delay the onset (i.e., the period prior toclinical manifestation of a disease) and/or reduce the risk ofdeveloping or worsening a disease. Within the meaning of the presentinvention, the term “treat” also encompasses preventing and/or reducinga positive symptom associated with schizophrenia or 22q11 DS, such as,e.g., hallucinations, delusions, disorganized thought, or psychosis.

As used herein the term “therapeutically effective” applied to dose oramount refers to that quantity of a compound or pharmaceuticalcomposition that is sufficient to result in a desired activity (e.g.,decrease in positive symptoms associated with schizophrenia and/or22q11DS) upon administration to a subject in need thereof. Note thatwhen a combination of active ingredients is administered, the effectiveamount of the combination may or may not include amounts of eachingredient that would have been effective if administered individually.The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the condition being treated, the particular drug or drugs employed,the mode of administration, and the like. An appropriate “effective”amount in any individual case may be determined by one of ordinary skillin the art using routine experimentation, based upon the informationprovided herein.

The phrase “pharmaceutically acceptable”, as used in connection withcompositions of the invention, refers to molecular entities and otheringredients of such compositions that are physiologically tolerable anddo not typically produce untoward reactions when administered to amammal (e.g., a human). Preferably, as used herein, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in mammals, and moreparticularly in humans.

As used herein, the term “combination” of a composition of the inventionand at least a second pharmaceutically active ingredient means at leasttwo, but any desired combination of compounds can be deliveredsimultaneously or sequentially (e.g., within a 24 hour period). It iscontemplated that when used to treat various diseases, the compositionsand methods of the present invention can be utilized with othertherapeutic methods/agents suitable for the same or similar diseases.Such other therapeutic methods/agents can be co-administered(simultaneously or sequentially) to generate additive or synergisticeffects. Suitable therapeutically effective dosages for each agent maybe lowered due to the additive action or synergy.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the compound is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussolution saline solutions and aqueous dextrose and glycerol solutionsare preferably employed as carriers, particularly for injectablesolutions. Alternatively, the carrier can be a solid dosage formcarrier, including but not limited to one or more of a binder (forcompressed pills), a glidant, an encapsulating agent, a flavorant, and acolorant. Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin.

An “individual” or “subject” or “animal”, as used herein, refers tohumans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs,etc.) and experimental animal models of schizophrenia or 22q11 DS. In apreferred embodiment, the subject is a human.

The term “associated with” is used to encompass any correlation,co-occurrence and any cause-and-effect relationship.

The term “about” means within an acceptable error range for theparticular value as determined by one of ordinary skill in the art,which will depend in part on how the value is measured or determined,i.e., the limitations of the measurement system. For example, “about”can mean within an acceptable standard deviation, per the practice inthe art. Alternatively, “about” can mean within an order of magnitude,preferably within 50%, more preferably within 20%, still more preferablywithin 10%, even more preferably within 5%, and most preferably within1% of a given value or range. Alternatively, particularly with respectto biological systems or processes, the term can mean within an order ofmagnitude, preferably within 2-fold, of a value. Where particular valuesare described in the application and claims, unless otherwise stated,the term “about” is implicit and in this context means within anacceptable error range for the particular value.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the content clearly dictates otherwise.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition. Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989 (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); Ausubel, F. M. et al.(eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc.,1994. These techniques include site directed mutagenesis as described inKunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985), U.S. Pat. No.5,071,743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360(1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh andGuengerich, BioTech. 24: 428-431 (1998); Ray and Nickoloff, BioTech. 13:342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang andMalcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641(1999), U.S. Pat. Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14.3: 74-75 (2001), U.S. Pat. Nos. 5,702,931, 5,780,270, and 6,242,222,Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson,Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996),Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch andJoly, Nucl. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J.Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993),Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al.,Meth. Molec. Biol. 67: 209-218.

Therapeutic Methods of the Invention

In one embodiment, the present invention provides a method for treatmentand/or prevention of schizophrenia in a subject in need thereof, themethod comprising administering to the subject a therapeuticallyeffective amount of (i) miR-338-3p or a mimic or a functional derivative(including functional fragments) thereof, or (ii) a vector expressingsaid miR-338-3p or mimic or functional derivative thereof, or (iii) anagent capable of increasing the level or activity of miR-338-3p. Inanother embodiment, the invention provides a method for treatment of22q11 deletion syndrome in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of (i) miR-338-3p or a mimic or a functional derivative thereof,or (ii) a vector expressing said miR-338-3p or mimic or functionalderivative thereof, or (iii) an agent capable of increasing the level oractivity of miR-338-3p. In yet another embodiment, the inventionprovides a method for treatment of a positive symptom of schizophreniain a subject in need thereof, the method comprising administering to thesubject a therapeutically effective amount of (i) miR-338-3p or a mimicor a functional derivative thereof, or (ii) a vector expressing saidmiR-338-3p or mimic or functional derivative thereof, or (iii) an agentcapable of increasing the level or activity of miR-338-3p. In oneembodiment, the miR-338-3p or mimic or derivative thereof targets Drd2,e.g., Drd2 present in the thalamus. In another embodiment, themiR-338-3p or mimic or derivative thereof mediates TC disruption, e.g.,by mediating the Dgcr8-Drd2 mechanism.

The therapeutic methods of the invention encompass over-expressingmiR-338-3p, functional derivatives thereof or miR-338-3p mimics, e.g.,using viral constructs, or using sense-based oligonucleotides ormodified-oligonucleotide mimics (e.g., technologies from miRNATherapeutics and miRagen Therapeutics), or inhibiting negative oractivating positive miRNA regulators (transcriptional or epigenetic),etc.

The miR-338-3p can be expressed from recombinant viral vectors. Therecombinant viral vectors of the invention comprise sequences encodingthe miR-338-3p and any suitable promoter for expressing the RNAsequences. Typical promoters for mammalian cell expression include,e.g., SV40 early promoter, CMV immediate early promoter (see, e.g., U.S.Pat. Nos. 5,168,062 and 5,385,839), mouse mammary tumor virus LTRpromoter, adenovirus major late promoter (Ad MLP), herpes simplex viruspromoter, murine metallothionein gene promoter, and U6 or H1 RNA pol IIIpromoter. Non-limiting examples of promoters useful for expressionmiRNA338-3p in the methods of the present invention include, e.g.,Synapsin promoter (neuron specific), CamKIIa promoter (specific forexcitatory neurons), CMV promoter, and β-actin promoter. Cell-type- ortissue-specific promoters can be used to express miRNA to allow for celltype- or tissue-specific expression. For example, the recombinant viralvectors of the invention can comprise inducible or regulatable promotersfor expression of the miR-338-3p in thalamic neuronal cells.

Any viral vector capable of accepting the coding sequences for themiR-338-3p can be used. For example, vectors derived from adenovirus(AV), adeno-associated virus (AAV), retroviruses (e.g., lentiviruses(LV), Rhabdoviruses, murine leukemia virus), Sindbis virus, herpesvirus, and the like. The tropism of the viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate. For example, lentiviral vectors of theinvention can be pseudotyped with surface proteins from vesicularstomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectorsof the invention can be made to specifically target certain cells ortissues by engineering the vectors to express certain capsid proteinserotypes. Currently, there are several AAV serotypes available that canbe used for tissue enrichment based on natural tropism toward specificcell types and interaction between different cellular receptors andserotypes. For example, an AAV vector expressing a serotype 2 capsid ona serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene inthe AAV 2/2 vector can be replaced by a serotype 5 capsid gene toproduce an AAV 2/5 vector. Techniques for constructing AAV vectors whichexpress different capsid protein serotypes are within the skill in theart; see, e.g., Rabinowitz J. E. et al. (2002), J Virol 76:791801. Amethod for constructing the recombinant AV vector, and a method fordelivering the vector into target cells, are also described in Xia etal. (2002), Nat. Biotech. 20:1006-1010. Suitable AAV vectors forexpressing the miRNAs, methods for constructing the recombinant AAVvector, and methods for delivering the vectors into target cells aredescribed in Samulski et al. (1987), J. Virol. 61:3096-3101; Fisher etal. (1996), J. Virol., 70:520-532; Samulski et al. (1989), J. Virol.63:3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International PatentApplication No. WO 94/13788; and International Patent Application No. WO93/24641.

Alternatively, the miR-338-3p can be expressed from recombinant circularor linear DNA plasmids using any suitable promoter, includinginducible/regulatable promoters. In one embodiment, the miR-338-3p isexpressed as RNA precursor molecules from a plasmid, and the precursormolecules are processed into the functional mature miR-338-3p by asuitable processing system, including, but not limited to, processingsystems existing within the thalamic neurons. Other suitable processingsystems include, e.g., the in vitro Drosophila cell lysate system (e.g.,as described in U.S. Published Patent Application No. 2002/0086356 toTuschl et al.) and the E. coli RNAse III system (e.g., as described inU.S. Published Patent Application No. 2004/0014113 to Yang et al.).

Selection of plasmids suitable for expressing miR-338-3p, methods forinserting nucleic acid sequences into the plasmid to express miR-338-3p,and methods of delivering the recombinant plasmid to the cells ofinterest are within the skill in the art. See, for example, Zeng et al.(2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol,20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi etal. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), GenesDev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; andPaul et al. (2002), Nat. Biotechnol. 20:505-508.

In one embodiment, a plasmid expressing the miR-338-3p comprises asequence encoding a miR precursor RNA under the control of theexcitatory neuron-specific promoter, such as CamKIIa. In anotherembodiment, a plasmid expressing the miR-338-3p comprises a sequenceencoding a miRNA precursor RNA under the control of the CMV and/or□-actin (ubiquitous) promoter. In yet another embodiment, a plasmidexpressing the miR-338-3p comprises a sequence encoding a miR precursorRNA under the control of a neuron specific Synapsin promoter.

In the therapeutic methods of the invention, miR-338-3p, mimics andfunctional derivatives thereof can be also administered directly. SuchmiR-338-3p, mimics and functional derivatives can be chemicallysynthesized or recombinantly produced using methods known in the art. Inone embodiment, miRNA are chemically synthesized using appropriatelyprotected ribonucleoside phosphoramidites and a conventional DNA/RNAsynthesizer. Commercial suppliers of synthetic RNA molecules orsynthesis reagents include, e.g., Proligo (Hamburg, Germany), DharmaconResearch (Lafayette, Colo., U.S.A.), Pierce Chemical (part of PerbioScience, Rockford, Ill., U.S.A.), Glen Research (Sterling, Va., U.S.A.),ChemGenes (Ashland, Mass., U.S.A.) and Cruachem (Glasgow, UK).

In some embodiments, of the invention, a synthetic miRNA contains one ormore design elements. These design elements include, but are not limitedto: (i) a replacement group for the phosphate or hydroxyl of thenucleotide at the 5′ terminus of the complementary region; (ii) one ormore sugar modifications. In certain embodiments, a synthetic miRNA hasa nucleotide at its 5′ end of the complementary region in which thephosphate and/or hydroxyl group has been replaced with another chemicalgroup (referred to as the “replacement design”). In some cases, thephosphate group is replaced, while in others, the hydroxyl group hasbeen replaced. In particular embodiments, the replacement group isbiotin, an amine group, a lower alkylamine group, an acetyl group,2′O-Me (2′oxygen-methyl), DMTO (4,4′-dimethoxytrityl with oxygen),fluorescein, a thiol, or acridine, though other replacement groups arewell known to those of skill in the art and can be used as well. Inparticular embodiments, the sugar modification is a 2′O-Me modification.In further embodiments, there is one or more sugar modifications in thefirst or last 2 to 4 residues of the complementary region or the firstor last 4 to 6 residues of the complementary region. In a particularembodiment, miR-338-3p, mimics and functional derivatives are maderesistant to degradation by nucleases, e.g., by incorporating one ormore ribonucleotides that are modified at the 2′-position with fluoro,amino, alkyl, alkoxy, and O-allyl.

Delivery of miR-338-3p, mimics and functional derivatives thereof can beenhanced by complexing with liposome nanoparticles, exosomes,polyethyleneimine, or atelocollagen (Rooij and Kauppinen, EMBO Mol Med.,2014, 6(7): 851-864).

Liposomes can increase the blood half-life of the nucleic acids.Liposomes suitable for use in the invention can be formed from standardvesicle-forming lipids, which generally include neutral or negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of factors such as thedesired liposome size and half-life of the liposomes in the bloodstream. A variety of methods are known for preparing liposomes, forexample, as described in Szoka et al. (1980), Ann. Rev. Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and5,019,369. The liposomes for use in the present methods can comprise aligand molecule that targets the liposome to thalamic neurons. Theliposomes for use in the present methods can also be modified so as toavoid clearance by the mononuclear macrophage system (“MIMS”) andreticuloendothelial system (“RES”). Such modified liposomes haveopsonization-inhibition moieties on the surface or incorporated into theliposome structure. In one embodiment, a liposome of the inventioncomprises both opsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer that significantly decreases the uptakeof the liposomes by the MMS and RES; e.g., as described in U.S. Pat. No.4,920,016. Opsonization inhibiting moieties suitable for modifyingliposomes are preferably water-soluble polymers with a number-averagemolecular weight from about 500 to about 40,000 daltons, and morepreferably from about 2,000 to about 20,000 daltons. Such polymersinclude polyethylene glycol (PEG) or polypropylene glycol (PPG)derivatives; e.g., methoxy PEG or PPG, and PEG or PPG stearate;synthetic polymers such as polyacrylamide or poly N-vinyl pyrrolidone;linear, branched, or dendrimeric polyamidoamines; polyacrylic acids;polyalcohols, e.g., polyvinylalcohol and polyxylitol to which carboxylicor amino groups are chemically linked, as well as gangliosides, such asganglioside GM1. Copolymers of PEG, methoxy PEG, or methoxy PPG, orderivatives thereof, are also suitable. In addition, the opsonizationinhibiting polymer can be a block copolymer of PEG and either apolyamino acid, polysaccharide, polyamidoamine, polyethyleneamine, orpolynucleotide. The opsonization inhibiting polymers can also be naturalpolysaccharides containing amino acids or carboxylic acids, e.g.,galacturonic acid, glucuronic acid, mannuronic acid, hyaluronic acid,pectic acid, neuraminic acid, alginic acid, carrageenan; animatedpolysaccharides or oligosaccharides (linear or branched); orcarboxylated polysaccharides or oligosaccharides, e.g., reacted withderivatives of carbonic acids with resultant linking of carboxylicgroups. Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes”.

The opsonization inhibiting moiety can be bound to the liposome membraneby any one of numerous well known techniques. For example, anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor, and then bound to amembrane. Similarly, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive animation usingNa(CN)BH3 and a solvent mixture, such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Liposomes modified with opsonization-inhibition moieties remain in thecirculation much longer than unmodified liposomes. For this reason, suchliposomes are sometimes called “stealth” liposomes. Stealth liposomesare known to accumulate in tissues fed by porous or “leaky”microvasculature. Thus, tissue characterized by such microvasculaturedefects will efficiently accumulate these liposomes; see Gabizon, et al.(1988), Proc. Natl. Acad. Sci., USA, 18:6949-53. In addition, thereduced uptake by the RES lowers the toxicity of stealth liposomes bypreventing significant accumulation of the liposomes in the liver andspleen.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated nucleic acids (Kaneda et al., 1989). In otherembodiments, the liposome may be complexed or employed in conjunctionwith nuclear non-histone chromosomal proteins (HMG-I) (Kato et al.,1991). In yet further embodiments, the liposome may be complexed oremployed in conjunction with both HVJ and HMG-I. In that such expressionconstructs have been successfully employed in transfer and expression ofnucleic acid in vitro and in vivo, then they are applicable for thepresent invention. Where a bacterial promoter is employed in the DNAconstruct, it also will be desirable to include within the liposome anappropriate bacterial polymerase.

Exosomes are nano-sized vesicles (30-120 nm in size) produced by manycell types, including dendritic cells (DC), B cells, T cells, mastcells, epithelial cells, and tumor cells. These vesicles are formed byinward budding of late endosomes and are then released to theextracellular environment upon fusion with the plasma membrane. Exosomescan be isolated from cells (e.g., by centrifugation) and loaded withmiRNA using, e.g., lipofectamine or electroporation.

Other expression constructs which can be employed to deliver miR-338-3por a mimic or a functional derivative thereof into cells arereceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis inalmost all eukaryotic cells. Because of the cell type-specificdistribution of various receptors, the delivery can be highly specific(Wu and Wu, 1993).

miR-338-3p or a mimic or a functional derivative thereof can be alsoadministered in combination with a cationic lipid. Examples of cationiclipids include, but are not limited to, lipofectin, DOTMA, DOPE, andDOTAP. The publication of WO/0071096 describes different formulations,such as a DOTAP:cholesterol or cholesterol derivative formulation thatcan effectively be used for gene therapy. Other disclosures also discussdifferent lipid or liposomal formulations including nanoparticles andmethods of administration; these include, but are not limited to, U.S.Patent Publication 20030203865, 20020150626, 20030032615, and20040048787. Methods used for forming particles are also disclosed inU.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901,6,200,801, and 5,972,900.

It is contemplated that when used to treat various diseases, thecompositions and methods of the present invention can be combined withother therapeutic agents suitable for the same or similar diseases.Also, two or more embodiments of the invention may be alsoco-administered to generate additive or synergistic effects. Whenco-administered with a second therapeutic agent, the embodiment of theinvention and the second therapeutic agent may be simultaneously orsequentially (in any order). Suitable therapeutically effective dosagesfor each agent may be lowered due to the additive action or synergy. Asa non-limiting example, the invention can be combined with othertherapies that decrease the positive symptoms of schizophrenia, e.g.,antipsychotics.

Compositions and Methods of Administration

The invention provides that miR-338-3p or a mimic or a functionalderivative thereof, or a vector expressing said miR-338-3p or mimic orfunctional derivative thereof, or an agent capable of increasing thelevel or activity of miR-338-3p can be administered to the subject toreplenish the endogenous miR-338-3p that is down-regulated in thalamicneurons of the subjects suffering from schizophrenia and/or 22q11 DS.The invention further provides that the isolated miR-338-3p or a mimicor a functional derivative thereof, or a vector expressing saidmiR-338-3p or mimic or functional derivative thereof, or an agentcapable of increasing the level or activity of miR-338-3p can be used aspharmaceutical compositions and can be optionally combined with otherantipsychotics, therapeutic molecules and/or treatments. In certainembodiments, miR-338-3p, or a mimic or a functional derivative thereof,or a vector expressing said miR-338-3p or mimic or functional derivativethereof, or an agent capable of increasing the level or activity ofmiR-338-3p, is used before, during, and after antipsychotics incombination therapies for treating positive symptoms associated withschizophrenia and/or 22q11 DS. The invention encompasses any now knownor later developed antipsychotics for treating schizophrenia and/or22q11 DS.

miR-338-3p molecules or mimics or functional derivatives thereof caninclude one or more modifications (e.g., to the base moiety, sugarmoiety, phosphate moiety, phosphate-sugar backbone, or a combinationthereof). For example, the phosphodiester linkages may be modified toinclude at least one heteroatom other than oxygen, such as nitrogen orsulfur. In this case, for example, the phosphodiester linkage may bereplaced by a phosphothioester linkage. Similarly, bases may be modifiedto block the activity of adenosine deaminase. Other examples of usefulmodifications are morpholino modifications and LNA. Where the miRNAmolecule is produced synthetically, or by in vitro transcription, amodified ribonucleoside may be introduced during synthesis ortranscription. Non-limiting examples of modified base moieties includeinosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurineNon-limiting examples of modified sugar moieties include arabinose,2-fluoroarabinose, xylulose, and hexose. Modified miRNAs may containsubstituted sugar moieties comprising one of the following at the 2′position: OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂ or O(CH₂)_(n)CH₃ where nis from 1 to about 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—; S—, or N-alkyl; O—, S—,or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted sialyl;a fluorescein moiety; a reporter group; a group for improving thepharmacokinetic properties; or a group for improving the pharmacodynamicproperties, and other substituents having similar properties. ModifiedmiRNAs may also have sugar mimetics such as cyclobutyls or othercarbocyclics in place of the pentofuranosyl group. Non-limiting examplesof modifications of phosphate backbone include a phosphorothioate, aphosphorodithioate, a phosphoramidothioate, a phosphoramidate, aphosphordiamidate, a methylphosphonate, a phosphotriester, an alkylphosphotriester, and a formacetal or analog thereof, as well as chimerasbetween methylphosphonate and phosphodiester, short chain alkyl, orcycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Specific non-limiting examples includethose with CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂, CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones (wherephosphodiester is O—PO₂—O—CH₂). U.S. Pat. No. 5,677,437 describesheteroaromatic oligonucleoside linkages. Nitrogen linkers or groupscontaining nitrogen can also be used to prepare oligonucleotide mimics(U.S. Pat. Nos. 5,792,844 and 5,783,682). U.S. Pat. No. 5,637,684describes phosphoramidate and phosphorothioamidate oligomeric compounds.Also envisioned are modified miRNA molecules having morpholino backbonestructures in which the bases are linked to 6-membered morpholine rings,which are connected to other morpholine-linked bases via non-ionicphosphorodiamidate intersubunit linkages. Morpholino miRNAs are highlyresistant to nucleases and have good targeting predictability (U.S. Pat.No. 5,034,506; Summerton, Biochim. Biophys. Acta 1999; 1489:141-158;Summerton and Weller, Antisense Nucleic Acid Drug Dev. 1997; 7:187-195;Arora et al., J. Pharmacol. Exp. Ther. 2000;292:921-928; Qin et al.,Antisense Nucleic Acid Drug Dev. 2000; 10:11-16; Heasman et al., Dev.Biol. 2000; 222:124-134; Nasevicius and Ekker, Nat. Genet. 2000;26:216-220). Another type of a useful modification is thepeptide-nucleic acid (PNA) backbone: the phosphodiester backbone of theoligonucleotide may be replaced with a polyamide backbone, the basesbeing bound directly or indirectly to the aza nitrogen atoms of thepolyamide backbone (Nielsen et al., Science 1991;254:1497). In otherembodiments, locked nucleic acids (LNA) can be used (reviewed in, e.g.,Jepsen and Wengel, Curr. Opin. Drug Discov. Devel. 2004; 7:188-194;Crinelli et al., Curr. Drug Targets 2004; 5:745-752). LNA are nucleicacid analog(s) with a 2′-O, 4′-C methylene bridge. This bridge restrictsthe flexibility of the ribofuranose ring and locks the structure into arigid C3-endo conformation, conferring enhanced hybridizationperformance and exceptional biostability.

Modified miRNAs can include appending groups such as, e.g., peptides, oragents facilitating transport across the cell membrane (see, e.g.,Letsinger et al., Proc. Natl. Acad. Sci. USA 1989; 86:6553-6556;Lemaitre et al., Proc. Natl. Acad. Sci. USA 1987; 84:648-652; PCTPublication No. WO 88/09810) or blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134), etc.

miR-338-3p or mimics or functional derivatives thereof used in thepresent invention can be synthesized by standard methods known in theart, e.g., by use of an automated synthesizer. Following chemicalsynthesis, miRNA molecules are deprotected and purified (e.g., by gelelectrophoresis or HPLC). Alternatively, standard procedures may usedfor in vitro transcription of miRNA from DNA templates carrying RNApolymerase promoter sequences (e.g., T7 or SP6 RNA polymerase promotersequences). See, e.g., Donzé and Picard, Nucleic Acids Res. 2002;30:e46; and Yu et al., Proc. Natl. Acad. Sci. USA 2002; 99:6047-6052.miRNA molecules may be also formed within a cell by transcription of RNAfrom an expression construct introduced into the cell. The expressionconstructs for in vivo production of miRNA molecules comprise miRNAencoding sequences operably linked to elements necessary for the propertranscription of the miRNA encoding sequence(s), including promoterelements and transcription termination signals. Preferred promoters foruse in such expression constructs include the polymerase-III HI-RNApromoter (see, e.g., Brummelkamp et al., supra) and the U6polymerase-III promoter (see, e.g., Sui et al., supra; Paul, et al.supra; and Yu et al., supra). The miRNA expression constructs canfurther comprise vector sequences that facilitate the cloning of theexpression constructs. Standard vectors that maybe used in practicingthe current invention are known in the art (e.g., pSilencer 2.0-U6vector, Ambion Inc., Austin, TX).

In some embodiments, miR-338-3p or a mimic or a functional derivativethereof, or a vector expressing said miR-338-3p or mimic or functionalderivative thereof, or an agent capable of increasing the level oractivity of miR-338-3p, is formulated into a suitable pharmaceuticalpreparation such as, e.g., solution, suspension, tablet, dispersibletablet, pill, capsule, powder, sustained release formulation or elixir,for oral administration; sterile solution or suspension for parenteraladministration; powdered or liquid spray, nose drops, a gel or ointmentfor intranasal administration; powdered or liquid spray foradministration by inhalation; films for sublingual administration; patchfor transdermal administration, etc. miR-338-3p or a mimic or afunctional derivative thereof, or a vector expressing said miR-338-3p ormimic or functional derivative thereof, or an agent capable ofincreasing the level or activity of miR-338-3p, can be formulated intopharmaceutical compositions using any of the techniques and proceduresknown in the art (see, e.g., Ansel Introduction to Pharmaceutical DosageForms, Fourth Edition 1985, 126).

In the compositions, effective concentrations of miR-338-3p or a mimicor a functional derivative thereof, or a vector expressing saidmiR-338-3p or mimic or functional derivative thereof, or an agentcapable of increasing the level or activity of miR-338-3p is (are) mixedwith a suitable pharmaceutical carrier or vehicle.

Pharmaceutically acceptable derivatives include acids, bases, enolethers and esters, salts, esters, hydrates, solvates and prodrug forms.A suitable derivative is selected such that its pharmacokineticproperties are superior with respect to at least one characteristic tothe corresponding parent agent. The miR-338-3p, or its mimics, may bederivatized prior to formulation.

In one embodiment of the invention, miR-338-3p or a mimic or afunctional derivative thereof, or a vector expressing said miR-338-3p ormimic or functional derivative thereof, or an agent capable ofincreasing the level or activity of miR-338-3p is administeredintranasally. Compositions for intranasal administration can compriseone or more nasal delivery-enhancing agents. As used herein, “nasaldelivery-enhancing agents” include agents which enhance the release orsolubility (e.g., from a formulation delivery vehicle), diffusion rate,penetration capacity and timing, uptake, residence time, stability,effective half-life, peak or sustained concentration levels, clearanceand other desired nasal delivery characteristics (e.g., as measured atthe site of delivery, or at a selected target site of activity such asthe brain) of miR-338-3p or a mimic or a functional derivative thereof,or a vector expressing said miR-338-3p or mimic or functional derivativethereof, or an agent capable of increasing the level or activity ofmiR-338-3p. Enhancement of mucosal delivery can thus occur by any of avariety of mechanisms, for example by increasing the diffusion,transport, persistence or stability of miR-338-3p or a mimic or afunctional derivative thereof, or a vector expressing said miR-338-3p ormimic or functional derivative thereof, or an agent capable ofincreasing the level or activity of miR-338-3p, enzyme inhibition,increasing membrane fluidity, modulating the availability or action ofcalcium and other ions that regulate intracellular or paracellularpermeation, solubilizing mucosal membrane components (e.g., lipids),changing non-protein and protein sulfhydryl levels in mucosal tissues,increasing water flux across the mucosal surface, modulating epithelialjunctional physiology, reducing the viscosity of mucus overlying themucosal epithelium, reducing mucociliary clearance rates, increasingnasal blood flow and other mechanisms. Suitable mucosal deliveryenhancing agents will be clear to a person skilled in the art ofpharmacology and arc further described hereafter.

The pharmaceutical compositions of the present invention can beadministered intranasally as a powdered or liquid spray, nose drops, agel or ointment, through a tube or catheter, by syringe, packtail,pledget or by submucosal infusion. The compositions for intranasaladministration can be simple aqueous (e.g., saline) solutions.Alternatively, they can contain various additional ingredients whichenhance stability and/or nasal delivery of miR-338-3p or a mimic or afunctional derivative thereof, or a vector expressing said miR-338-3p ormimic or functional derivative thereof, or an agent capable ofincreasing the level or activity of miR-338-3p. Such additionalingredients are well known in the art. Non-limiting examples of usefuladditional ingredients for enhancing nasal delivery include, e.g., (a)aggregation inhibitory agents (e.g., polyethylene glycol, dextran,diethylaminoethyl dextran, and carboxy methyl cellulose), (b) chargemodifying agents, (c) pH control agents, (d) degradative enzymeinhibitors (e.g., amastatin and bestatin [see, e.g., O'Hagan et al.,Pharm. Res. 1990, 7: 772-776 and WO 05/120551]; pegylation with PEGmolecules, preferably low molecular weight PEG molecules [e.g. 2 kDa;Lee et al., Calcif Tissue Int. 2003, 73 : 545-549]); (c) mucolytic ormucus clearing agents (e.g., n-acetyl-cysteine, propylgallate andcysteine methionine dimmers, chaotropes [see, e.g., WO 04/093917]), (f)ciliostatic agents; (g) membrane penetration-enhancing agents, (h)modulatory agents of epithelial junction physiology, such as nitricoxide (NO) stimulators, chitosan, and chitosan derivatives; (i)vasodilator agents, (j) selective transport-enhancing agents, and (k)stabilizing delivery vehicles, carriers, supports or complex-formingagents. See, e.g., EP 037943, EP 094157, EP 173990, EP 214898, EP215697, EP 327756, EP 490806, U.S. Pat. Nos. 4,476,116, 5,759,565, WO04/093917 and WO 05/120551.

The activity or physical stability of miR-338-3p or a mimic or afunctional derivative thereof, or a vector expressing said miR-338-3p ormimic or functional derivative thereof, or an agent capable ofincreasing the level or activity of miR-338-3p in aqueous solutions orlyophilized preparations can be enhanced by various additives such as,e.g., polyols (including sugars [e.g., sucrose and Ficoll 70]), aminoacids, and various salts. For example, miR-338-3p or a mimic or afunctional derivative thereof microparticles can be prepared by simplylyophilizing or spray drying a solution containing various stabilizingadditives described above. A wide non-limiting range of suitable methodsand anti-aggregation agents are available for incorporation within thecompositions of the invention such as disclosed in WO 05/120551, Breslowet al. (J. Am. Chcm. Soc. 1996; 118: 11678-11681), Breslow et al. (PNASUSA 1997; 94: 11156-11158), Breslow et al. (Tetrahedron Lett. 1998;2887-2890), Zutsh i et al. (Curr. Opin. Chem. Biol. 1998; 2: 62-66).Daugherty et al. (J. Am. Chem. Soc. 1999; 121: 4325-4333), Zutshi et al.(J. Am. Chem. Soc. 1997; 119: 484 -4845), Ghosh et al. (Chem. Biol.1997; 5: 439-445), Hamuro et al. (Angew. Chem. Int. Fd. Fngl. 1997; 36:2680-2683), Alberg et al., Science 1993; 262: 248-250), Tauton et al.(J. Am. Chem. Soc. 1996; 118: 10412-10422), Park et al. (J. Am. Chem.Soc. 1999; 121: 8-13), Prasanna ct al. (Biochemistry 1998;37:6883-6893), Tiley et al. (J. Am. Chem. Soc. 1997; 119: 7589-7590),Judice et al. (PNAS USA 1997; 94: 13426-13430), Fan et al. (J. Am. Chem.Soc. 1998; 120: 8893-8894), Gamboni et al. (Biochemistry 1998; 37:12189-12194).

Non-limiting examples of membrane penetration-enhancing agents useful inthe intranasal compositions of the invention include, e.g., (i) asurfactant (e.g., Tween 80, Poloxamer 188, polysorbates; see also EP490806, U.S. Pat. No. 5,759,565, and WO 04/093917), (ii) a bile salt orbile salt derivative (e.g., unsaturated cyclic ureas and Transcutol).(iii) a phospholipid or fatty acid additive, mixed micelle, liposome, orcarrier, (iv) an alcohol, (v) an enamine, (vi) a nitric oxide donorcompound (e.g., S-nitroso-N-acetyl-DF-penicillamine, NOR 1, NOR4, whichare preferably co-administered with an NO scavenger such as carboxy-PITOor doclofenac sodium), (vii) a long-chain amphipathic molecule (e.g.,dcacylmethyl sulfoxide, azone, sodium lauryl sulfate, oleic acid) (viii)a small hydrophobic penetration enhancer, (ix) sodium salicylate or asalicylic acid derivative (e.g., acetyl salicylate, chol ine salicylate,salicylamide, etc.), (x) a glycerol ester of acetoacetic acid, (xi) acyclodcxtrin or bcta-cyclodextrin derivative, (xii) a medium-chain fattyacid including mono- and diglycerides (e.g., sodium caprate—extracts ofcoconut oil, Capmul), (xiii) a chelating agent (e.g., citric acid,salicylates), (xiv) an amino acid or salt thereof (e.g.monoaminocarboxlic acids such as glycine, alanine, phenylalanine, proline, hydroxyproline, etc.; hydroxyamino acids such as serine; acidicamino acids such as aspartic acid, glutamic acid, etc; and basic aminoacids such as lysine etc., inclusive of their alkali metal or alkalineearth metal salts), (xv) an N-acctylamino acid or salt (thereof, (xvi)an enzyme degradative to a selected membrane component, (xvii) aninhibitor of fatty acid synthesis, (xviii) an inhibitor of cholesterolsynthesis, (xix) cationic polymers, or any combination thereof. Themembrane penetration-enhancing agent can be also selected from smallhydrophilic molecules, including but not limited to, dimethyl sulfoxide(DMSO), dimethylformamide, ethanol, propylene glycol, and the2-pyrrolidones. Additional membrane penetration enhancers includeemulsifiers (e.g. sodium oleyl phosphate, sodium lauryl phosphate,sodium lauryl sulfate, sodium myristyl sulfate, polyoxyethylene alkylethers, polyoxyethylencalkyl esters, etc.), caproic acid, lactic acid,malic acid and citric acid and alkali metal salts thereof,pyrrolidonecarboxylic acids, alkylpyrrolidonecarboxylic acid esters,N-alkylpyrrolidones, proline acyl esters, and the like; mixed micelles;glycerol esters of acetoacetic acid (e.g., glyceryl-1,3-diacetoacetateor 1,2-isopropylideneglycerine-3-acetoacetate) and triglycerides (e.g.,amylodextrin, Estaram 299, Miglyol 810); cyclodcxtrins and(3-cyclodextrin derivatives (e.g., 2-hydroxypropyl-p-cyclodextrin andheptakis (2,6-di-0-methyl-[3-cyclodextrin) which can be optionallyformulated in an oleaginous base; and N-acetylamino acids(N-acetylalaninc, N-acetylphenylalaninc, N-acetylserine,N-acetylglycine, N-acetyllysine, N-acetylglutamic acid, N-acetylproline,N-acetylhydroxyproline, etc.) and their salts (alkali metal salts andalkaline earth metal salts), as well as other penetration-promotingagents that are physiologically compatible for intranasal delivery. See,e.g., WO04/093917, WO05/120551 and Davis and Ilium (Clin. Pharmacokinet.2003, 42: 1107-1128).

Non-limiting examples of useful absorption enhancers include, e.g.,surfactants, glycosides, cyclodextrin and glycols. Non-limiting examplesof useful bioadhesive agents include, e.g., carbopol, cellulose agents,starch, dextran, and chitosan.

In various embodiments of the invention, miR-338-3p or a mimic or afunctional derivative thereof, or a vector expressing said miR-338-3p ormimic or functional derivative thereof, or an agent capable ofincreasing the level or activity of miR-338-3p is combined with one ormore of the nasal delivery-enhancing agents recited above. These nasaldelivery-enhancing agents may be admixed, alone or together, with thenasal carrier and with miR-338-3p or a mimic or a functional derivativethereof, or a vector expressing said miR-338-3p or mimic or functionalderivative thereof, or an agent capable of increasing the level oractivity of miR-338-3p, or otherwise combined therewith in apharmaceutically acceptable formulation or delivery vehicle. For nasaldelivery-enhancing agents to be of value within the invention, it isgenerally desired that any significant changes in permeability of themucosa be reversible within a time frame appropriate to the desiredduration of drug delivery. Furthermore, there should be no substantial,cumulative toxicity, nor any permanent deleterious changes induced inthe barrier properties of the nasal mucosa with long term use.

The useful delivery volume of the intranasal pharmaceutical compositionsof the invention is limited by the size of the nasal cavity. Suitabledelivery volumes will be clear to a person skilled in the art ofpharmacology. Preferably, the total composition quantity administered ateach nasal appl ication comprises from about 0.02 to 0.5 ml, preferablyabout 0.07 to 0.3 ml, typically about 0.09-0.1 ml. A solid compositionmay comprise from 1 to 30 mg carrier per dosage, more particularly 4 to20 mg.

The liquid compositions of the invention may be prepared by bringinginto intimate admixture miR-338-3p or a mimic or a functional derivativethereof, or a vector expressing said miR-338-3p or mimic or functionalderivative thereof, or an agent capable of increasing the level oractivity of miR-338-3p in the liquid carrier optionally together withthe further ingredients, additives and/or agents. Preferably theresulting mixture is then lyophilized and dissolved in water or aqueoussaline for use in a liquid form according to the invention. The solidnasal composition of the invention may be prepared in conventionalmanner. miR-338-3p or a mimic or a functional derivative thereof, or avector expressing said miR-338-3p or mimic or functional derivativethereof, or an agent capable of increasing the level or activity ofmiR-338-3p may be admixed with the carrier particles, e.g., a polymerbase or cellulose product in conventional manner, optionally withfurther ingredients, additives and/or agents as indicated above e.g. amucosal delivery enhancing agent or surfactant such as disclosed.miR-338-3p or a mimic or a functional derivative thereof, or a vectorexpressing said miR-338-3p or mimic or functional derivative thereof, oran agent capable of increasing the level or activity of miR-338-3p maybe in solution, e.g., an aqueous or alcoholic solution when being mixedwith the carrier particles and the solvent evaporated, e.g., underfreeze-drying or spray drying. Such drying may be effected under theconventional conditions. Alternatively, the mixture may be compacted orgranulated and then be pulverized and/or sieved. If desired theparticles may be coated. According to a preferred embodiment of theinvention, the nasal composition is prepared by lyophilisation. Ahomogeneous solution, preferably aqueous, containing miR-338-3p or amimic or a functional derivative thereof, or a vector expressing saidmiR-338-3p or mimic or functional derivative thereof, or an agentcapable of increasing the level or activity of miR-338-3p and optionallycontaining further ingredients, additives and/or agents as discussedabove, is prepared and then submitted to lyophilisation in analogy withknown lyophilisation procedures, and to subsequent drying. The resultingpowder may then be dissolved in a liquid excipient or nasal carrierbefore administration, e.g., to reconstitute nasal drops, gel or spray.Alternatively it may be administered as such in the form of lyophilizedpowder or it may be mixed with further ingredients, additives and/oragents as discussed above. For example, a lyophilized powder comprisingmiR-338-3p or a mimic or a functional derivative thereof, or a vectorexpressing said miR-338-3p or mimic or functional derivative thereof, oran agent capable of increasing the level or activity of miR-338-3p butfree of any nasal carrier may be prepared and then admixed with thedesired nasal carrier or mixture of nasal carriers.

The present invention encompasses any delivery device that is suitablefor nasal administration of the compositions of the invention.Preferably, such means administers a metered dosage of the composition.The composition of the present invention may be packed in anyappropriate form or container as long as a means is provided to deliverthe composition to the nasal mucosa. Non-limiting examples of usefulintranasal delivery devices include, e.g., instillation catheters,droppers, unit-dose containers, squeeze bottles pump sprays, airless andpreservative-free sprays, compressed air nebulizers, metered-discinhalers, insufflators and pressurized metered dose inhalers.

For administration of a liquid in drop form, compositions of theinvention can be placed in a container provided with a conventionaldropper/closure device, e.g., comprising a pipette or the like,preferably delivering a substantially fixed volume of composition/drop.

For administration of an aqueous solution as a nasal spray, the aqueoussolution may be dispensed in spray form by a variety of methods known tothose skilled in the art. For example, such compositions will be put upin an appropriate atomising device, e.g. in a pump-atomiser, or thelike. The atomising device will be provided with appropriate means, suchas a spray adaptor for delivery of the aqueous spray to the naris.Preferably it will be provided with means ensuring delivery of asubstantially fixed volume of composition/actuation (i.e. perspray-unit). Examples of nasal sprays include nasal actuators producedby Ing. Krich Pfeiffer GmbH, Radolfzell, Germany (see U.S. Pat. Nos.4,511,069, 4,778,810, 5,203,840, 5,860,567, 5,893,484, 6.227,415, and6,364,166. Additional aerosol delivery forms may include, e.g.compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers.

Alternatively the spray may be bottled under pressure in an aerosoldevice. The propellant may be a gas or a liquid (e.g. a fluorinatedand/or chlorinated hydrocarbon). The spray composition may be suspendedor dissolved in a liquid propellant. Stabilizing and/or suspendingagents and/or co-solvents may be present.

A dry powder may be readily dispersed in an inhalation device asdescribed in U.S. Pat. No. 6,514,496 and Garcia-Arieta et al., Biol.Pharm. Bull. 2001; 24: 1411-1416.

If desired a powder or liquid may be filled into a soft or hard capsuleor in a single dose device adapted for nasal administration. The powdermay be sieved before filled into the capsules such as gelatine capsules.The delivery device may have means to break open the capsule. Thepowdery nasal composition can be directly used as a powder for a unitdosage form. The contents of the capsule or single dose device may beadministered using e.g. an insufflator. Preferably it will be providedwith means ensuring dosing of a substantially fixed amount ofcomposition.

Delivery devices are important not only for delivering miR-338-3p or amimic or a functional derivative thereof, or a vector expressing saidmiR-338-3p or mimic or functional derivative thereof, or an agentcapable of increasing the level or activity of miR-338-3p, but also forproviding an appropriate environment for storage. This would includeprotection from microbial contamination and chemical degradation. Thedevice and formulation should be compatible so as to avoid potentialleaching or adsorption.

The delivery device (or its packaging) can be optionally provided with alabel and/or with instructions for use indicating that the compositionshould be used intranasally.

In another embodiment, the composition of the invention can be providedas a nasal insert having miR-338-3p or a mimic or a functionalderivative thereof, or a vector expressing said miR-338-3p or mimic orfunctional derivative thereof, or an agent capable of increasing thelevel or activity of miR-338-3p dispersed therein. The insert may beretained in the naris, but flushed by the nasal mucus, and may bedesigned to release miR-338-3p or a mimic or a functional derivativethereof, or a vector expressing said miR-338-3p or mimic or functionalderivative thereof, or an agent capable of increasing the level oractivity of miR-338-3p at the same place in the naris. Suitable nasalinsert types include nasal plugs, tampons and the like, further examplesof nasal inserts, their characteristics and preparation are described inHP 490806.

The compounds and compositions of the invention may further compriseagents, which facilitate delivery across the blood brain barrier (BBB).Non-limiting examples of such useful agents include, e.g., animplantable reservoir (Omaya reservoir), polysialation, functionalizednanocarriers (e.g., nanoparticles coated with transferrin or transferrinreceptor [TR] antibodies), exosomes, liposomes (e.g., liposomes coatedwith targeting molecules such as antibodies, Trojan Horses Liposomes[THL]), antibodies (e.g., antibodies against transferrin receptor [TR]or insulin receptor [HIR], BBB transmigrating Llama single domainantibodies (sdAb)), chimeric peptides (e.g., Angiopeps derived fromproteins expressing the Kunitz domain), low-density lipoprotein receptorrelated proteins 1 and 2 (LRP-1 and 2), diphtheria toxin receptor (DTR),mesenchyme stem cells, receptor-associated protein, apolipoprotein E,melanotransferrin/p97, etc.

In one embodiment, in order to enhance brain delivery of miR-338-3p or amimic or a functional derivative thereof, or a vector expressing saidmiR-338-3p or mimic or functional derivative thereof, or an agentcapable of increasing the level or activity of miR-338-3p, the patientis treated in a manner so as to increase the selective permeability ofthe blood-brain barrier (BBB). Treatments to selectively increase thepermeability of the BBB in a patient include, but are not limited to,the administration of about 1 to about 1000 μg/kg body weight,preferably about 10 to about 100 μg/kg body weight, of IGF-I (e.g., as abolus injection to a patient about 0.5 to 10 hours, preferably about 1hour, before the inhibitor administration).

The amount of miR-338-3p or a mimic or a functional derivative thereof,or a vector expressing said miR-338-3p or mimic or functional derivativethereof, or an agent capable of increasing the level or activity ofmiR-338-3p, administered and the regimen of administration depends onabsorption, inactivation and excretion rates of the active agent, thephysicochemical characteristics of the agent, the severity of thecondition to be alleviated, the age, condition, body weight, sex anddiet of the patient, the disease state, other medications administered,and other factors known to those of skill in the art. An effectiveamount to treat the disease would broadly range (e.g., between about0.001 mg and about 2000 mg per kg body weight of the recipient per day),and may be administered as a single dose or divided doses.

It is to be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions.

The compositions of the invention are intended to be administered by asuitable route, including by way of example and without limitationorally, parenterally (e.g., intravenously, subcutaneously,intramuscularly), intranasally, by inhalation, sublingually, andtopically. miR-338-3p or a mimic or a functional derivative thereof, ora vector expressing said miR-338-3p or mimic or functional derivativethereof, or an agent capable of increasing the level or activity ofmiR-338-3p, can be administered to a subject by any suitable enteral orparenteral administration route. Suitable enteral administration routesfor the present methods include, e.g., oral, rectal, or intranasaldelivery. Suitable parenteral administration routes include, e.g.,intravascular administration (e.g., intravenous bolus injection,intravenous infusion, intra-arterial bolus injection, intra-arterialinfusion and catheter instillation into the vasculature); peri- andintra-tissue injection; subcutaneous injection or deposition, includingsubcutaneous infusion (such as by osmotic pumps); direct application tothe tissue of interest, for example by a catheter or other placementdevice (e.g., a retinal pellet or a suppository or an implant comprisinga porous, non-porous, or gelatinous material); and inhalation.Particularly suitable administration routes are injection, infusion anddirect injection into the brain and/or within thalamic neurons), and/orvia viral vector (e.g., AAV and/or lentiviral vector) mediated delivery.

The compositions are in liquid, semi-liquid or solid form and areformulated in a manner suitable for each route of administration.

Solutions or suspensions can include any of the following components, inany combination: a sterile diluent, including by way of example withoutlimitation, water for injection, saline solution, fixed oil,polyethylene glycol, glycerine, propylene glycol or other syntheticsolvent; antimicrobial agents, such as benzyl alcohol and methylparabens; antioxidants, such as ascorbic acid and sodium bisulfite;chelating agents, such as ethylenediaminetetraacetic acid (EDTA);buffers, such as acetates, citrates and phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose.

In instances in which the agents exhibit insufficient solubility,methods for solubilizing agents may be used. Such methods are known tothose of skill in this art, and include, but are not limited to, usingco-solvents, such as, e.g., dimethylsulfoxide (DMSO), using surfactants,such as TWEEN®80, or dissolution in aqueous sodium bicarbonate.Pharmaceutically acceptable derivatives of the agents may also be usedin formulating effective pharmaceutical compositions.

The composition can contain along with the active agent, for example andwithout limitation: a diluent such as lactose, sucrose, dicalciumphosphate, or carboxymethylcellulose; a lubricant, such as magnesiumstearate, calcium stearate and talc; and a binder such as starch,natural gums, such as gum acacia gelatin, glucose, molasses,polyvinylpyrrolidone, celluloses and derivatives thereof, povidone,crospovidones and other such binders known to those of skill in the art.Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an active agentas defined above and optional pharmaceutical adjuvants in a carrier,such as, by way of example and without limitation, water, saline,aqueous dextrose, glycerol, glycols, ethanol, and the like, to therebyform a solution or suspension. If desired, the pharmaceuticalcomposition to be administered may also contain minor amounts ofnontoxic auxiliary substances such as wetting agents, emulsifyingagents, or solubilizing agents, pH buffering agents and the like, suchas, by way of example and without limitation, acetate, sodium citrate,cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodiumacetate, triethanolamine oleate, and other such agents. Actual methodsof preparing such dosage forms are known, or will be apparent, to thoseskilled in this art (e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., 15th Edition, 1975). The composition orformulation to be administered will, in any event, contain a quantity ofthe active agent in an amount sufficient to alleviate the symptoms ofthe treated subject.

The active agents or pharmaceutically acceptable derivatives may beprepared with carriers that protect the agent against rapid eliminationfrom the body, such as time release formulations or coatings. Thecompositions may include other active agents to obtain desiredcombinations of properties.

Oral pharmaceutical dosage forms include, by way of example and withoutlimitation, solid, gel and liquid. Solid dosage forms include tablets,capsules, granules, and bulk powders. Oral tablets include compressed,chewable lozenges and tablets which may be enteric-coated, sugar-coatedor film-coated. Capsules may be hard or soft gelatin capsules, whilegranules and powders may be provided in non-effervescent or effervescentform with the combination of other ingredients known to those skilled inthe art.

In certain embodiments, the formulations are solid dosage forms, such ascapsules or tablets. The tablets, pills, capsules, troches and the likecan contain any of the following ingredients, or agents of a similarnature: a binder; a diluent; a disintegrating agent; a lubricant; aglidant; a sweetening agent; and a flavoring agent.

Examples of binders include, by way of example and without limitation,microcrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, sucrose, and starch paste. Lubricantsinclude, by way of example and without limitation, talc, starch,magnesium or calcium stearate, lycopodium and stearic acid. Diluentsinclude, by way of example and without limitation, lactose, sucrose,starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidantsinclude, by way of example and without limitation, colloidal silicondioxide. Disintegrating agents include, by way of example and withoutlimitation, crosscarmellose sodium, sodium starch glycolate, alginicacid, corn starch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Coloring agents include, by way of example andwithout limitation, any of the approved certified water soluble FD and Cdyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include, by way of example andwithout limitation, sucrose, lactose, mannitol and artificial sweeteningagents such as saccharin, and any number of spray dried flavors.Flavoring agents include, by way of example and without limitation,natural flavors extracted from plants such as fruits and syntheticblends of agents which produce a pleasant sensation, such as, but notlimited to peppermint and methyl salicylate. Wetting agents include, byway of example and without limitation, propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylenelaural ether. Emetic-coatings include, by way of example and withoutlimitation, fatty acids, fats, waxes, shellac, ammoniated shellac andcellulose acetate phthalates. Film coatings include, by way of exampleand without limitation, hydroxyethylcellulose, sodiumcarboxymethylcellulose, polyethylene glycol 4000 and cellulose acetatephthalate.

If oral administration is desired, the agent could be provided in acomposition that protects it from the acidic environment of the stomach.For example, the composition can be formulated in an enteric coatingthat maintains its integrity in the stomach and releases the activeagent in the intestine. The composition may also be formulated incombination with an antacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier such as fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The agents can also be administered as acomponent of an elixir, suspension, syrup, wafer, sprinkle, chewing gumor the like. A syrup may contain, in addition to the active agents,sucrose as a sweetening agent and certain preservatives, dyes andcolorings and flavors. The active materials can also be mixed with otheractive materials which do not impair the desired action, or withmaterials that supplement the desired action, such as antacids, H2blockers, and diuretics.

Pharmaceutically acceptable carriers included in tablets are binders,lubricants, diluents, disintegrating agents, coloring agents, flavoringagents, and wetting agents. Enteric-coated tablets, because of theenteric-coating, resist the action of stomach acid and dissolve ordisintegrate in the neutral or alkaline intestines. Sugar-coated tabletsare compressed tablets to which different layers of pharmaceuticallyacceptable substances are applied. Film-coated tablets are compressedtablets which have been coated with a polymer or other suitable coating.Multiple compressed tablets are compressed tablets made by more than onecompression cycle utilizing the pharmaceutically acceptable substancespreviously mentioned. Coloring agents may also be used in the abovedosage forms. Flavoring and sweetening agents are used in compressedtablets, sugar-coated, multiple compressed and chewable tablets.Flavoring and sweetening agents are useful in the formation of chewabletablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions,suspensions, solutions and/or suspensions reconstituted fromnon-effervescent granules and effervescent preparations reconstitutedfrom effervescent granules. Aqueous solutions include, for example,elixirs and syrups. Emulsions are either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wetting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents may be used in any of theabove dosage forms.

Solvents include, by way of example and without limitation, glycerin,sorbitol, ethyl alcohol and syrup. Examples of preservatives include,without limitation, glycerin, methyl and propylparaben, benzoic acid,sodium benzoate and alcohol. Non-aqueous liquids utilized in emulsionsinclude, by way of example and without limitation, mineral oil andcottonseed oil. Emulsifying agents include, by way of example andwithout limitation, gelatin, acacia, tragacanth, bentonite, andsurfactants such as polyoxyethylene sorbitan monooleate. Suspendingagents include, by way of example and without limitation, sodiumcarboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Diluentsinclude, by way of example and without limitation, lactose and sucrose.Sweetening agents include, by way of example and without limitation,sucrose, syrups, glycerin and artificial sweetening agents such assaccharin. Wetting agents include, by way of example and withoutlimitation, propylene glycol monostearate, sorbitan monooleate,diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organicacids include, by way of example and without limitation, citric andtartaric acid. Sources of carbon dioxide include, by way of example andwithout limitation, sodium bicarbonate and sodium carbonate. Coloringagents include, by way of example and without limitation, any of theapproved certified water soluble FD and C dyes, and mixtures thereof.Flavoring agents include, by way of example and without limitation,natural flavors extracted from plants such fruits, and synthetic blendsof agents which produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for examplepropylene carbonate, vegetable oils or triglycerides, is encapsulated ina gelatin capsule. Such solutions, and the preparation and encapsulationthereof, are disclosed in U.S. Pat. Nos. 4,328,245, 4,409,239, and4,410,545. For a liquid dosage form, the solution (e.g., in apolyethylene glycol) may be diluted with a sufficient quantity of apharmaceutically acceptable liquid carrier (e.g., water) to be easilymeasured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active agent or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. RE28819 and U.S. Pat. No.4,358,603. Briefly, such formulations include, but are not limited to,those containing an agent provided herein, a dialkylated mono- orpoly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

Tablets and capsules formulations may be coated as known by those ofskill in the art in order to modify or sustain dissolution of the activeingredient. Thus, for example and without limitation, they may be coatedwith a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

Parenteral administration generally characterized by injection, eithersubcutaneously, intramuscularly or intravenously, is also contemplatedherein. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients include, by way of example and without limitation, water,saline, dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as, for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (e.g., U.S. Pat. No. 3,710,795)is also contemplated herein. Briefly, an inhibitor of Nt5e or MR isdispersed in a solid inner matrix (e.g., polymethylmethacrylate,polybutylmethacrylate, plasticized or unplasticized polyvinylchloride,plasticized nylon, plasticized polyethyleneterephthalate, naturalrubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene,ethylene-vinylacetate copolymers, silicone rubbers,polydimethylsiloxanes, silicone carbonate copolymers, hydrophilicpolymers such as hydrogels of esters of acrylic and methacrylic acid,collagen, cross-linked polyvinylalcohol and cross-linked partiallyhydrolyzed polyvinyl acetate) that is surrounded by an outer polymericmembrane (e.g., polyethylene, polypropylene, ethylene/propylenecopolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetatecopolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber,chlorinated polyethylene, polyvinylchloride, vinylchloride copolymerswith vinyl acetate, vinylidene chloride, ethylene and propylene, ionomerpolyethylene terephthalate, butyl rubber epichlorohydrin rubbers,ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcoholterpolymer, and ethylene/vinyloxyethanol copolymer) that is insoluble inbody fluids. The agent diffuses through the outer polymeric membrane ina release rate controlling step. The percentage of active agentcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the agent and theneeds of the subject.

Lyophilized powders can be reconstituted for administration assolutions, emulsions, and other mixtures or formulated as solids orgels. The sterile, lyophilized powder is prepared by dissolving an agentprovided herein, or a pharmaceutically acceptable derivative thereof, ina suitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at,typically, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. Generally,the resulting solution will be apportioned into vials forlyophilization. Each vial will contain, by way of example and withoutlimitation, a single dosage (10-1000 mg, such as 100-500 mg) or multipledosages of the agent. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, about 1-50 mg, such as about 5-35 mg, for example, about9-30 mg of lyophilized powder, is added per mL of sterile water or othersuitable carrier. The precise amount depends upon the selected agent.Such amount can be empirically determined.

miR-338-3p or a mimic or a functional derivative thereof, or a vectorexpressing said miR-338-3p or mimic or functional derivative thereof, oran agent capable of increasing the level or activity of miR-338-3p maybe formulated as aerosols for application e.g., by inhalation orintranasally (e.g., as described in U.S. Pat. Nos. 4,044,126, 4,414,209,and 4,364,923). These formulations can be in the form of an aerosol orsolution for a nebulizer, or as a microtine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, by way of example andwithout limitation, have diameters of less than about 50 microns, suchas less than about 10 microns.

The agents may be also formulated for local or topical application, suchas for application to the skin and mucous membranes (e.g.,intranasally), in the form of nasal solutions, gels, creams, andlotions.

Other routes of administration, such as transdermal patches are alsocontemplated herein. Transdermal patches, including iotophoretic andelectrophoretic devices, are well known to those of skill in the art.For example, such patches are disclosed in U.S. Pat. Nos. 6,267,983,6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317,5,983,134, 5,948,433, and 5,860,957.

miR-338-3p or a mimic or a functional derivative thereof, or a vectorexpressing said miR-338-3p or mimic or functional derivative thereof, oran agent capable of increasing the level or activity of miR-338-3p maybe packaged as articles of manufacture containing packaging material anda label that indicates that miR-338-3p or a mimic or a functionalderivative thereof, or a vector expressing said miR-338-3p or mimic orfunctional derivative thereof, or an agent capable of increasing thelevel or activity of miR-338-3p, or pharmaceutically acceptablederivative thereof, are used for replenish miR-338-3p in thalamicneurons for rescue abnormal function of thalamic neurons and theirabnormal sensitivity to antipsychotics, so as to treat one or morepositive symptoms of schizophrenia and/or 22q11 DS.

In one embodiment of any of the above compositions, the compositionfurther comprises an inhibitor of Drd2, including an inhibitor of theexpression of Drd2, now known or later discovered. In one embodiment ofany of the above compositions, the composition further comprises anactivator of Dcgr8, including an activator of the expression of Dcgr8,now known or later discovered.

Diagnostic Methods of the Invention

In one embodiment, the invention provides a method for determiningefficacy of a treatment for schizophrenia or 22q11 deletion syndrome ina subject, the method comprising:

(a) determining the level of miR-338-3p in thalamic neurons or in abodily fluid sample obtained from the subject before the treatment,

(b) determining the level of miR-338-3p in thalamic neurons of thesubject after the treatment,

(c) comparing the levels determined in steps (a) and (b), and

(d) determining that the treatment is effective if the level ofmiR-338-3p in thalamic neurons or in the bodily fluid sample obtainedfrom the subject has increased after the treatment.

In another embodiment, the invention provides a method for determiningthe likelihood of developing a positive symptom of schizophrenia in asubject, the method comprising:

(a) determining the level of miR-338-3p in thalamic neurons or in abodily fluid sample obtained from the subject,

(b) comparing the level determined in step (a) to a control level, and

(c) determining that the subject is at risk of developing positivesymptoms of schizophrenia if the level of miR-338-3p in thalamic neuronsor in the bodily fluid sample obtained from the subject is lower thanthe control level.

In the diagnostic methods of the invention, the level of miR-388-3p ismeasured in a biological sample obtained from the subject. For example,a brain tissue sample can be removed from the subject, and neurons canbe isolated by standard techniques. Alternatively, a bodily fluid samplecan be used. Non-limiting examples of useful bodily fluids fordetermination of the levels of miR-338-3p include, e.g., blood, urine,saliva, CSF. The blood sample may comprise whole blood, bloodlymphocytes, peripheral blood mononuclear cells (PBMCs), blood plasma,or blood serum. The identification of miRNA expression in the bloodsample will typically take place ex vivo, but the present invention alsocontemplates in vivo testing.

A corresponding control sample can be obtained from a healthy age- andgender-matched subject or a population of such healthy subjects. Thecontrol sample is then processed along with the sample from the subject,so that the levels of miR-338-3p in the subject's sample can be comparedto the corresponding miR-338-3p levels from the control sample. Areference miRNA expression standard for the biological sample can alsobe used as a control.

The level of miR-338-3p in a sample can be measured using any techniquethat is suitable for detecting RNA levels in a biological sample.Suitable techniques include hybridization (e.g., Northern blot analysis,in situ hybridization), array-based assays, PCR-based assays, andsequencing. Array-based assays include, e.g., commercial arrays fromAgilent, Exiqon, Affymetrix or custom-designed two-color arrays with acommon reference (e.g., a specific quantity of ‘artificial’ miRNA forall probes on the chip or a specific sample such as, e.g., large batchesof RNA isolated from patient blood, etc), or solution hybridizationassays such as Ambion mirVana miRNA Detection Kit). Sequencing methodsinclude, e.g., direct sequencing by one of the next generationsequencing technologies (e.g., Helicos small RNA sequencing, miRNABeadArray (Illumina), Roche 454 (FLX-Titanium), and ABI SOLiD). Forreview of additional applicable techniques see, e.g., Chen et al., BMCGenomics, 2009, 10:407; Kong et al., J Cell Physiol. 2009; 218:22-25.

In a particular embodiment, the level of at least one miR-338-3p isdetected using Northern blot analysis. For example, total cellular RNAcan be purified from cells by homogenization in the presence of nucleicacid extraction buffer, followed by centrifugation. Nucleic acids areprecipitated, and DNA is removed by treatment with DNase andprecipitation. The RNA molecules are then separated by gelelectrophoresis on agarose gels according to standard techniques, andtransferred to nitrocellulose filters. The RNA is then immobilized onthe filters by heating. Detection and quantification of specific RNA isaccomplished using appropriately labeled DNA or RNA probes complementaryto the RNA in question. See, for example, Molecular Cloning: ALaboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold SpringHarbor Laboratory Press, 1989, Chapter 7.

Suitable probes for Northern blot hybridization of miR-338-3p can beproduced from the nucleic acid sequences provided in the figures andinclude, but are not limited to, probes having at least about 70%, 75%,80%, 85%, 90%, 95%, 98%, 99% or complete complementarity to miR-338-3p.Methods for preparation of labeled DNA and RNA probes, and theconditions for hybridization thereof to target nucleotide sequences, aredescribed in Molecular Cloning: A Laboratory Manual, J. Sambrook et al.,eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters10 and 11.

For example, the nucleic acid probe can be labeled with, e.g., aradionuclide, such as ³H, ³²P, ³³P, ¹⁴C, or ³⁵S; a heavy metal; a ligandcapable of functioning as a specific binding pair member for a labeledligand (e.g., biotin, avidin or an antibody); a fluorescent molecule; achemiluminescent molecule; an enzyme or the like.

Where radionuclide labeling of DNA or RNA probes is not practical, therandom-primer method can be used to incorporate an analogue, forexample, the dTTP analogue5-(N-(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridinetriphosphate, into the probe molecule. The biotinylyated probeoligonucleotide can be detected by reaction with biotin-bindingproteins, such as avidin, streptavidin, and antibodies (e.g.,anti-biotin antibodies) coupled to fluorescent dyes or enzymes thatproduce color reactions.

In addition to Northern and other RNA hybridization techniques,determining the levels of RNA transcripts can be accomplished using thetechnique of in situ hybridization. This technique requires fewer cellsthan the Northern blotting technique, and involves depositing wholecells onto a microscope cover slip and probing the nucleic acid contentof the cell with a solution containing radioactive or otherwise labelednucleic acid (e.g., cDNA or RNA) probes. This technique is particularlywell-suited for analyzing tissue biopsy samples from subjects. Suitableprobes for in situ hybridization of a given miRNA can be produced fromthe nucleic acid sequences, and include, but are not limited to, probeshaving at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or completecomplementarity to a miRNA of interest, as described above.

The relative number of miRNA molecules in cells can also be determinedby reverse transcription of miRNA, followed by amplification of thereverse-transcribed transcripts by polymerase chain reaction (RT-PCR).Non-limiting examples of useful commercial RT-PCR assays include TaqmanmiRNA assays (stem-loop assays; Applied Biosystems) and LNA-based miRNAPCR assays (poly-A-based assays; Exiqon) or quantitative RT-PCR basedarray method (qPCR-array). Other methods of amplification include ligasechain reaction (LCR), transcription-mediated amplification (TMA), stranddisplacement amplification (SDA) and nucleic acid sequence basedamplification (NASBA).

The levels of miRNA can be quantified in comparison with an internalstandard, for example, the level of mRNA from a “housekeeping” genepresent in the same sample. A suitable “housekeeping” gene for use as aninternal standard includes, e.g., myosin or glyceraldehyde-3-phosphatedehydrogenase (G3PDH). Methods for performing quantitative andsemi-quantitative RT-PCR, and variations thereof, are well known tothose of skill in the art.

Useful methods of miRNA isolation and purification, include, e.g.,Qiazol or Trizol extraction or the use of commercial kits (e.g.,miRNeasy kit [Qiagen], MirVana RNA isolation kit [Ambion/ABI], miRACLE[Agilent], High Pure miRNA isolation kit [Roche], and miRNA Purificationkit [Norgen Biotek Corp.]), concentration and purification onanion-exchangers, magnetic beads covered by RNA-binding substances, oradsorption of certain miRNA on complementary oligonucleotides.

In some embodiments, miRNA degradation in patients' samples is reducedor eliminated. Useful methods for reducing or eliminating miRNAdegradation include, without limitation, adding RNase inhibitors (e.g.,RNasin Plus [Promega], SUPERase-In [ABI], etc.), use of guanidinechloride, guanidine isothiocyanate, N-lauroylsarcosine, sodium dodecylsulphate (SDS), or a combination thereof. Air-exposure-related RNAdegradation can be reduced, e.g., by storage of samples in an inert airenvironment, performing RNA extraction within 3-4 days from the time ofsample collection to minimize air-related RNA degradation, or minimizingtime to tissue fixation. Reducing miRNA degradation in samples isparticularly important when sample storage and transportation isrequired prior to miRNA quantification.

In one embodiment of any of the above methods, the method furthercomprises determining the level of Dcgr8 and/or Drd2 expression, such asfor example and not limitation, by hybridization (including protein andnucleic acid hybridization assays, e.g., ELISA, Western blotting,Northern blotting, Southern blotting), array-based assays, PCR-basedassays (e.g., qPCR), and sequencing.

Kits of the Invention

In conjunction with the above diagnostic methods, the present inventionalso provides various kits comprising primers and/or probes specific formiR-338-3p. The kits of the invention can be useful, e.g., fordiagnosing schizophrenia or for determining efficacy of a treatment forschizophrenia or 22q11 deletion syndrome.

A kit of the invention can also provide reagents for primer extensionand amplification reactions. For example, in some embodiments, the kitmay further include one or more of the following components: a reversetranscriptase enzyme, a DNA polymerase enzyme (such as, e.g., athermostable DNA polymerase), a polymerase chain reaction buffer, areverse transcription buffer, and deoxynucleoside triphosphates (dNTPs).Alternatively (or in addition), a kit can include reagents forperforming a hybridization assay. The detecting agents can includenucleotide analogs and/or a labeling moiety, e.g., directly detectablemoiety such as a fluorophore (fluorochrome) or a radioactive isotope, orindirectly detectable moiety, such as a member of a binding pair, suchas biotin, or an enzyme capable of catalyzing a non-soluble colorimetricor luminometric reaction. In addition, the kit may further include atleast one container containing reagents for detection of electrophoresednucleic acids. Such reagents include those which directly detect nucleicacids, such as fluorescent intercalating agent or silver stainingreagents, or those reagents directed at detecting labeled nucleic acids,such as, but not limited to, ECL reagents. A kit can further includemiRNA isolation or purification means as well as positive and negativecontrols. A kit can also include a notice associated therewith in a formprescribed by a governmental agency regulating the manufacture, use orsale of diagnostic kits. Detailed instructions for use, storage andtroubleshooting may also be provided with the kit. A kit can also beoptionally provided in a suitable housing that is preferably useful forrobotic handling in a high throughput setting.

The components of the kit may be provided as dried powder(s). Whenreagents and/or components are provided as a dry powder, the powder canbe reconstituted by the addition of a suitable solvent. It is envisionedthat the solvent may also be provided in another container. Thecontainer will generally include at least one vial, test tube, flask,bottle, syringe, and/or other container means, into which the solvent isplaced, optionally aliquoted. The kits may also comprise a secondcontainer means for containing a sterile, pharmaceutically acceptablebuffer and/or other solvent.

Where there is more than one component in the kit, the kit also willgenerally contain a second, third, or other additional container intowhich the additional components may be separately placed. However,various combinations of components may be comprised in a container.

Such kits may also include components that preserve or maintain DNA orRNA, such as reagents that protect against nucleic acid degradation.Such components may be nuclease or RNase-free or protect against RNases,for example. Any of the compositions or reagents described herein may becomponents in a kit.

EXAMPLES

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

Example 1: Materials and Methods

Animals. Mice of both sexes were used for all experiments. Df(16)1/+ andDgcr8^(+/−) mouse strains were reported previously^(40, 68) and wereback-crossed onto the C57BL/6J genetic background for at least 10generations. The miR-338^(+/−) and miR-338^(−/−) mice were generatedfrom embryonic stem cells from C57BL/6N-A^(tmlBrd) mice that werepurchased from the Mutant Mouse Regional Resource Center (MMRRC; clone#034476-UCD). C57BL/6 blastocyst injections were performed by theTransgenic/Gene Knockout Shared Resource at St. Jude Children's ResearchHospital (St. Jude). Chimeric mice were genotyped according to MMRRCprotocols by using the following primers: 5′ common reverse(ATAGCATACATTATACGAAGTTATCACTGG; SEQ ID NO: 39), 5′ gene-specific(CTTCACTACACTCTCCCTAGTACAGTCTC; SEQ ID NO: 40), 3′ common forward(TCTAGAAAGTATAGGAACTTCCATGGTC; SEQ ID NO: 41), and 3′ gene-specific(AGGAGACTCATAGTTCTCTGTATCATAGC SEQ ID NO: 42). PCR was performed underthe following conditions: 93° C. for 3 min, 93° C. for 15 s, and 68° C.for 9 min for 8 cycles and then 93° C. for 15 s, 60° C. for 30 s, and68° C. for 9 min for 32 cycles. The mutant allele generated a 6.1-kbband with 5′ common-reverse and 5′ gene-specific primers and a 4.1-kbband with 3′ common-forward and 3′ gene-specific primers. The wild-type(WT) allele did not generate a band with either primer set. Subsequentgenotyping was performed at Transnetyx (Cordova, Tenn.). For themajority of experiments, mice were divided into groups according togenotype or viral injections, and the experimenters were blinded to thegenotype or treatment. The care and use of animals were reviewed andapproved by the St. Jude Institutional Animal Care and Use Committee.

Whole-Cell Electrophysiology. Acute primary thalamocortical (TC) slices(400-μm thick) containing the left auditory cortex (ACx) and the leftventral part of the medial geniculate nuclei (MGv) of the thalamus wereprepared as previously described⁵. Briefly, mouse brains were quicklyremoved and placed in cold (4° C.) dissecting artificial cerebrospinalfluid (ACSF) containing 125 mM choline-Cl, 2.5 mM KCl, 0.4 mM CaCl₂, 6mM MgCl₂, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, and 20 mM glucose (300-310mOsm), with 95% O₂/5% CO₂. Primary TC slices were obtained from the lefthemisphere by using a slicing angle of 15°. After a 1-h incubation inACSF [125 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 1.25 mM NaH₂PO₄,26 mM NaHCO₃, 20 mM glucose (300-310 mOsm), with 95% O₂/5% CO₂] at roomtemperature, the slices were transferred into the recording chamber andsuperfused (2-3 mL/min) with warm (30-32° C.) ACSF.

Whole-cell recordings were obtained from cell bodies of layer (L) 3/4thalamorecipient neurons in the ACx and thalamic-relay neurons in theMGv. Mice were chosen in a pseudorandom order, without theexperimenter's prior knowledge of genotype or treatments. Patch pipettes(open-pipette resistance, 3.5-5 MΩ) were filled with an internalsolution containing 125 mM CsMeSO₃, 2 mM CsCl, 10 mM HEPES, 0.1 mM EGTA,4 mM MgATP, 0.3 mM NaGTP, 10 mM Na₂ creatine phosphate, 5 mM QX-314, 5mM tetraethylammonium Cl (pH 7.4 adjusted with CsOH, 290-295 mOsm).Voltage-clamp recordings were made using a Multiclamp 700B (MolecularDevices, Sunnyvale Calif.), digitized (10 kHz), and recorded using thepCLAMP 10.0 software (Molecular Devices, Sunnyvale Calif.). EPSCs wererecorded at holding membrane potentials of −70 mV. In all experiments,membrane potentials were corrected for a liquid junction potential of−10 mV. TC excitatory postsynaptic currents (EPSCs) were evoked bycurrent pulses (duration, 100 μs) delivered to the thalamic radiationvia tungsten bipolar electrodes. Paired-pulse ratio (PPR) of TC andcorticocortical (CC) EPSCs and the NMDAR/AMPAR ratio were measured asdescribed previously³⁹. To ensure consistent access resistance of therecording electrode during long-term experiments, the peak amplitude ofa brief (10-ms) hyperpolarizing test pulse (−5 mV) was monitored, whichwas given 250 ms after a stimulus. Access resistance (Ra) in recordedneurons was typically 10 to 25 MΩ. Recordings were discarded if theaccess resistance was higher than 25 MΩ, or if it changed more than 15%during the course of the whole-cell recording.

Two-Photon Imaging. Two-photon laser-scanning microscopy was performedusing an Ultima imaging system, a Ti:sapphire Chameleon Ultrafemtosecond-pulsed laser, and 60×(0.9 NA) water-immersion infraredobjectives (Coherent, Santa Clara, Calif.). Synaptically evoked calciumtransients were measured in dendritic spines, the site of thalamicinputs, as described previously⁸¹. Briefly, Alexa Fluor 594 (30 μM) andFluo-5F (300 μM) (ThermoFischer Scientific) were included in theinternal pipette solution (see above) and were excited at 820 nm.Synaptically evoked changes in fluorescence of both fluorophores weremeasured in the line-scan mode (750 Hz) in spine heads and the parentdendritic shaft. Line scans were analyzed as changes in green (G,Fluo-5F) fluorescence normalized to red (R, Alexa Fluor 594)fluorescence (ΔG/R). The amplitude and probability of calcium transientswere measured in response to 10 to 20 stimulations delivered at 0.1 Hzto the thalamic radiation. Distance (angular) of the active thalamicinputs from the center of the soma was calculated usingmaximum-intensity projections of z-scan images of the entire cellcollected at lower magnification.

Optogenetics. In optogenetic experiments, the light-activated cationchannel ChR2 was expressed in the MGv by using adeno-associated virus(AAV) and optically induced EPSCs were evoked by briefly illuminating TCslices with a 473-nm light¹¹⁶. AAVs were generated from thepAAV-CaMKIIα-hChR2(H134R)-YFP-WPRE-pA (CamKIIα-ChR2-YFP) plasmid andproduced commercially (UNC Vector; serotype 2/1; 4×10¹² IFU/mL). AAVswere injected into the MGv as described previously⁷⁹. Adult mice wereanesthetized with isoflurane in pure oxygen, and a 200- to 400-nL sampleof virus was slowly pressure-injected into the MGv (from the bregma:anterior-posterior, −3.0 mm; medial-lateral, ±2.0 mm; dorsal-ventral,3.1 mm). Approximately 21 to 28 days after virus injection, the micewere decapitated, and TC slices were prepared. Confocal imaging of YFPin the MGv was used to verify on-target infection of CamKIIα-ChR2-YFPviruses. Short light pulses (10-200 mW) from a 473-nm laser weredirected to the slices through the visible light photoactivation moduleor through the objective.

Slice electrophysiology. Acute primary thalamocortical (TC) slices (400μm thick) containing the auditory cortex (ACx) and the ventral part ofthe medial geniculate nuclei (MGv) of the thalamus were prepared aspreviously described⁶. Briefly, mouse brains were quickly removed andplaced in cold (4° C.) dissecting artificial cerebrospinal fluid (ACSF)containing 125 mM choline-Cl, 2.5 mM KCl, 0.4 mM CaCl₂, 6 mM MgCl₂, 1.25mM NaH₂PO₄, 26 mM NaHCO₃, and 20 mM glucose (300-310 mOsm), with 95%O₂/5% CO₂. Primary TC slices were obtained from the left hemisphere byusing a slicing angle of 15°. After a 1 hour incubation in ACSF (125 mMNaCl, 2.5 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃,20 mM glucose [300-310 mOsm], with 95% O₂/5% CO₂) at room temperature,the slices were transferred into the recording chamber and superfused(2-3 mL/min) with warm (30-32° C.) ACSF.

Whole-cell recordings were obtained from cell bodies of layer (L) 3/4thalamorecipient neurons in the ACx and thalamic-relay neurons in theMGv. Patch pipettes (open pipette resistance, 3.5-5 MΩ) were filled withan internal solution containing 125 mM CsMeSO₃, 2 mM CsCl, 10 mM HEPES,0.1 mM EGTA, 4 mM MgATP, 0.3 mM NaGTP, 10 mM Na₂ creatine phosphate, 5mM QX-314, 5 mM tetraethylammonium Cl (pH 7.4 was adjusted with CsOH,290-295 mOsm). Voltage-clamp recordings were made using a Multiclamp700B, digitized (10 kHz), and recorded using the pCLAMP 10.0 software.EPSCs were recorded at holding membrane potentials of −70 mV. In allexperiments, membrane potentials were corrected for a liquid junctionpotential of −10 mV. TC EPSCs were evoked by current pulses (duration,100 μs) delivered to the thalamic radiation via tungsten bipolarelectrodes. To ensure consistent access resistance of the recordingelectrode during long-term experiments, the peak amplitude of a brief(10-ms) hyperpolarizing test pulse (−5 mV) was monitored given 250 msafter a stimulus. Access resistance (Ra) in recorded neurons wastypically 10 to 25 MΩ. Recordings were discarded if access resistancewas higher than 25 MΩ or if access resistance changed more than 15%during the course of the whole-cell recording.

miRNA microarray. Total RNA was isolated from 2- and 4-month-old maleWT, Df(16)1/+, and Dgcr8^(+/−) thalami containing MGv using mirVana RNAisolation kit (Life Technologies, Carlsbad, Calif.). Total RNAs (100 ng)were labeled using miRNA Complete Labeling and Hyb Kit (Agilent, SantaClara, Calif.), followed by hybridizing to the Mouse miRNA v19microarray (Agilent-046065) that contains 3,105 unique biologicalfeatured probes targeting 1,247 mature miRNAs according to mouse miRBaseversion 19.0 (www.mirbase.org; August 2012). Microarrays were scanned byusing an Agilent array scanner (G2565CA) at 3-μm resolution. Microarraydata were extracted by Agilent Feature Extraction software (v.10.5.1.1).The data process was performed using Partek software (St. Louis, Mo.).

After quantile normalization among arrays, each probe was summarized byaveraging with a single normalized intensity value. The Student's t-testwas used to determine statistical significance between sets ofreplicates from different experimental groups. The miRNA was consideredsignificantly differentially expressed if the p-value<0.01 and loge foldchange (FC)>0.2 for more than one probe targeting the mature form of themiRNA. The mRNAs targeted by differentially expressed miRNAs werepredicted using bioinformatics tools miRWalk⁷⁶ and TargetScan(www.targetscan.org).

Quantitative RT-PCR. Total RNA was isolated from various brain regions(i.e., the auditory thalamus containing the MGv, hippocampus, or cortex)by using mirVana RNA isolation Kit (Life Technologies, Carlsbad,Calif.). The synthesis of cDNA from mRNA was performed with iScript(Bio-Rad, Hercules, Calif.), and miRNA First-Strand cDNA Synthesis Kit(Agilent) was used to synthesize cDNA from miRNA. The experiments wereperformed using SYBR Green (Life Technologies, Carlsbad, Calif.). Thefollowing forward primers were used for miRNA analysis: mmu-miR-338-3p(TCCAGCATCAGTGATTTTGTTG, SEQ ID NO: 2), hsa-miR-338-3p(TCCAGCATCAGTGATTTTGTTG; SEQ ID NO: 44) mmu-miR-335-3p(TTTTTCATTATTGCTCCTGACC, SEQ ID NO: 3), mmu-miR-335-5p (TCAAGAGCAATAACGAAAAATGT, SEQ ID NO: 4), mmu-miR-337-3p (TCAGCTCCTATATGATGCCTTT, SEQ ID NO: 5), mmu-miR-337-5p (CGGCGTCATGCAGGAGTTGATT, SEQ ID NO:6), mmu-miR-3065-5p (TCAACAAAATCACTGATGCTGG, SEQ ID NO: 7), andmmu-miR-3065-3p (TCAGCACCAGGATATTGTTGGGGm SEQ ID NO: 8).

The universal reverse-primer specific to the sequence tag (miRNAFirst-Strand cDNA Synthesis Kit; Agilent) was used. The followingprimers were used for mRNA analysis: Drd2 forward(GGATGTCATGATGTGCACAGC, SEQ ID NO: 9), Drd2 reverse (CGCTTGCGGAGAACGATG,SEQ ID NO: 10), Aatk forward (ATGCTGGCCTGCCTGTGTTGT, SEQ ID NO: 11), andAatk reverse (AGGGGCAGGACATACACATCGG, SEQ ID NO: 12). The followingloading controls were used: U6 snRNA forward (CGCTTCGGCAGCACATATAC, SEQID NO: 13), U6 snRNA reverse (TTCACGAATTTGCGTGTCAT, SEQ ID NO: 14),SnoRNA202 (CTTTTGAACCCTTTTCCATCTG, SEQ ID NO: 15), and SnoRNA234(TTAACAAAAATTCGTCACTACCA, SEQ ID NO: 16). The same universal reverseprimer was used for SnoRNA202 and SnoRNA234. The same U6 snRNA primerswere used for human and mouse samples. Samples from each mouse were runin triplicate.

Human brain tissue. Postmortem samples of human MGv and ACx (Brodmannarea 41) were obtained from The Maryland Brain Collection (MarylandPsychiatric Research Center, University of Maryland School of Medicine,Catonsville, MD). The level of mature miR-338-3p wase tested in sixpatients with schizophrenia and six age-, race-, and sex-matched healthycontrols. Only samples with RNA integrity number>7 were used in theseexperiments (Agilent RNA 6000 Nano kit). The mean postmortem intervalwas 15.8±1.8 h for patients with schizophrenia and 16.5±1.2 h (p>0.05)for healthy controls. Quantitative RT-PCR for each brain tissue samplewas run in triplicate.

Plasmids and viruses. To overexpress miRNAs of interest, recombinantAAVs (serotype 5) were generated by cloning chimeric hairpins of themiRNAs of interest³¹ with hsa-miR-30a into the 3′ UTR of GFP under theCamKIla promoter using a previously described strategy³¹. The followingprimers were used: miR-338-3p-1 (GTACAGCTGTTGACAGTGAGCGACTCCAGCATCAGTGATTTTGTTGTGTGAA, SEQ ID NO: 17), miR-338-3p-2(CCATCTGTGGCTTCACACAACAAAATCACTGATGCTGGAGTCGCTCACTGTCAACA GCT, SEQ IDNO: 18), miR-338-3p-3 (GCCACAGATGGCAACAAAATCTGATGCTGGAGCTGCCTACTGCCTCGGAA, SEQ ID NO: 19), miR-338-3p-4 (AGCTTTCCGAGGCAGTAGGCAGCTCCAGCATCAGATTTTGTTG, SEQ ID NO: 20), miR-337-3p-1 (GTACAGCTGTTGACAGTGAGCGACTCAGCTCCTATATGATGCCTTTTGTGAA, SEQ ID NO: 21),miR-337-3p-2 (CCATCTGTGGCTTCACAAAAGGCATCATATAGGAGCTGAGTCGCTCACTGTCAACAGCT, SEQ ID NO: 22), miR-337-3p-3 (GCCACAGATGGAAAGGCATCATAGGAGCTGAGCTGCCTACTGCCTCGGAA, SEQ ID NO: 23), miR-337-3p-4 (AGCTTTCCGAGGCAGTAGGCAGCTCAGCTCCTATGATGCCTTT, SEQ ID NO: 24), miR-337-5p-1 (GTACAGCTGTTGACAGTGAGCGACCGGCGTCATGCAGGAGTTGATTTGTGAA, SEQ ID NO: 25),miR-337-5p-2 (CCATCTGTGGCTTCACAAATCAACTCCTGCATGACGCCGGTCGCTCACTGTCAACAGCT, SEQ ID NO: 26), miR-337-5p-3 (GCCACAGATGGAATCAACTCGCATGACGCCGGCTGCCTACTGCCTCGGAA, SEQ ID NO: 27), miR-337-5p-4(AGCTTTCCGAGGCAGTAGGCAGCCGGCGTCATGCGAGTTGATT, SEQ ID NO: 28),miR-335-3p-1 (GTACAGCTGTTGACAGTGAGCGACTTTTTCATTATTGCTCCTGACCTGT GAA, SEQID NO: 29), miR-335-3p-2 (CCATCTGTGGCTTCACAGGTCAGGAGCAATAATGAAAAAGTCGCTCACTGTCAACAGCT, SEQ ID NO: 30), miR-335-3p-3(GCCACAGATGGGGTCAGGAGATAATGAAAAAGCTGCCTACTGCCTCGGAA, SEQ ID NO: 31),miR-335-3p-4 (AGCTTTCCGAGGCAGTAGGCAGCTTTTTCATTATCTCCTG ACC, SEQ ID NO:32), miR-335-5p-1 (GTACAGCTGTTGACAGTGAGCGACTCAAGAGCAATAACGAAAAATGTTGTGAA, SEQ ID NO: 33), miR-335-5p-2 (CCATCTGTGGCTTCACAACATTTTTCGTTATTGCTCTTGAGTCGCTCACTGTCAACAGCT, SEQ ID NO: 34),miR-335-5p-3 (GCCACAGATGGACATTTTTCGATTGCTCTTGAGCTGCCTACT GCCTCGGAA, SEQID NO: 35), and miR-335-5p-4 (AGCTTTCCGAGGCAGTAGGCAGCTCAAGAGCAATCGAAAAATGT, SEQ ID NO: 36).

Generation of miR-338-3p sponges and Renilla and Firefly activity usingthe dual-luciferase reporter assay was performed as describedpreviously^(32,33). Twelve copies of the following sequences wereinserted for the miR-338-3p sponge (CAACAAAATGCGGATGCTGGA, SEQ ID NO:37) or scrambled control (GACACTGTGAGCGAAGACATA, SEQ ID NO: 38) into the3′ UTR of GFP under the control of the CamKIIα promoter. RecombinantAAVs (1-2×10¹³⁻¹⁴ particles/mL) were generated at the St. Jude VectorDevelopment & Production Core and injected into the MGvs of anesthetizedmice, as described previously⁶.

In the luciferase assay, multiple copies of the miR-338-3p sponge andscramble control were cloned into 3′-UTR of Renilla luciferase genecontained within the psiCHECK-2 vector (Promega). The plasmids weretransfected into HEK 293T cells along with miR-338-3p overexpressionplasmid or control pcDNA3.1 or irrelevant miR-185-5p overexpressingplasmid. After two days in culture, Renilla and Firefly activities weremeasured using the dual-luciferase reporter assay (Promega) according tothe manufacturer's instructions. The Renilla luciferase expression wasnormalized to Firefly luciferase expression as a readout.

Generation of miR-338 KO mice. Heterozygous embryonic stem cells fromC57BL/6N-A^(tm1Brd) mice were purchased from Mutant Mouse RegionalResource Center (clone #034476-UCD). C57BL/6 blastocysts injections wereperformed by the Transgenic/Gene Knockout Shared Resource at St. JudeChildren's Research Hospital. Genotyping of chimeric mice was performedaccording to Mutant Mouse Regional Recourse Center protocols using thefollowing primers: 5′ common reverse (ATAGCATACATTATACGAAGTTATCACTGG,SEQ ID NO: 39), 5′ gene-specific (CTTCACTACACTCTCCCTAGTACAGTCTC, SEQ IDNO: 40), 3′ common forward (TCTAGAAAGTATAGGAACTTCCATGGTC, SEQ ID NO:41), and 3′ gene-specific (AGGAGACTCATAGTTCTCTGTATCATAGC, SEQ ID NO:42). PCR conditions were a follows: 93° C. for 3 min, 93° C. for 15 s,and 68° C. for 9 min for 8 cycles and then 93° C. for 15 s, 60° C. for30 s, 68° C. for 9 min for 32 cycles. Mutant allele generates a6.1-kilobase band with 5′ common reverse and 5′ gene-specific primers,and 4.1-kilobase band with 3′ common forward and 3′ gene-specificprimers. WT allele does not generate a band with either primer set.Subsequent genotyping was performed at Transnetyx (Cordova, Tenn.).

Mouse behavioral tests. Prepulse (PPI) experiments were performed aspreviously described⁶. Briefly, each day before testing, the mice weretransported from the animal-housing room and allowed a 1-h habituationperiod in the testing room. Before experiments were initiated, the micewere allowed to acclimate to the Plexiglas restraint chamber (6 cm×6cm×4.8 cm) for 20 min. The mice then had a 5-min acclimation period to a65-dB background white noise, which played throughout the session. ForPPI experiments, three acoustic startles (8 kHz, 120 dB, 40 ms) weredelivered separated by a 15-s intertrial interval. The testing sessionconsisted of the following trials: pulse-alone, in which the startlepulse was presented; the combination of a 40-ms white-noise prepulse (74dB, 82 dB, or 90 dB) in WT and Dgcr8^(+/−) littermates and (70 dB, 80dB, or 90 dB) in WT and miR-338^(+/−) littermates and preceding thestartle pulse by 100 ms, and no stimuli. Trials were separated by 15 sand presented in a pseudo-random order. PPI was calculated as follows:100×(pulse-alone response−prepulse+pulse response)/pulse-alone response.

Auditory Brainstem Responses (ABR) experiments were performed aspreviously described^(34,86). Briefly, mice were anesthetized withAvertin (0.6 mg/g bodyweight, i.p.) and ABR was measured using a TuckerDavis Technology (TDT) System III with RZ6 Multiprocessor and BioSigRZsoftware (Tucker Davis Technology, Alachua, Fla.). Sounds were deliveredvia the MF-1 speaker in the open-field configuration. ABR waveforms wererecorded using sub-dermal needles placed at the vertex of the skull,below the pinna of the ear, and at the base of the tail. The needleswere connected to a low impedance head-stage (RA4LI, TDT) and fed intothe RZ6 multiprocessor through a pre-amplifier (RA4PA, Gain 20x, TDT)(Tucker Davis Technology, Alachua, Fla.). ABR waveforms were averagesobtained from 500 presentations of a tone (21 tones/s) in thealternating phase and were band-pass filtered (300 Hz -3 kHz). The ABRthreshold was defined as the minimum sound intensity that elicited awave above the noise level. All ABR experiments were conducted in asound booth (Industrial Acoustic Company, IAC, Model 120A double wall).

Statistical analyses. Data are represented as the means±SEM. Allstatistics were computed using the Sigma Plot software. Differences inmean data were determined by the t-test, Wilcoxon signed rank test, aone-way ANOVA followed by Student-Newman-Keuls post-hoc test, or atwo-way ANOVA, followed by Holm-Sidak multiple comparisons and wereconsidered significant at p<0.05.

Example 2: Delayed Disruption of TC Synaptic Transmission in 22q11DSmodels: The Sensitivity of TC Projections and TC Excitatory PostsynapticCurrents (EPSCs) Measurements in Animal Models at Different PostnatalAges

In adult (4- to 5-month-old) Df(16)1/+ murine models^(7,15) of 22q11DS(FIG. 1a ), a genetic disorder causing schizophrenia in approximately30% of patients^(10,16), Dgcr8 haploinsufficiency causes deficientsynaptic transmission in TC projections to the ACx⁶, a brain region thatis implicated in auditory hallucinations¹⁷⁻¹⁹. This deficiency ismediated by an aberrant elevation of Drd2 in the thalamic-relay neurons,which renders them abnormally sensitive to antipsychotics (Drd2antagonists)⁶.

Because the positive symptoms of schizophrenia arise during lateadolescence or early adulthood, the sensitivity of TC projections to theantipsychotic agent haloperidol was compared in Df(16)1/+ mice (FIG. 1a) and their wild-type (WT) littermates at different postnatal ages,ranging from 1.5 to 7 months. Using single-cell recordings, TCexcitatory postsynaptic currents (EPSCs) were measured from layer (L)3/4 pyramidal neurons, the main thalamorecipient neurons in the ACx²⁰,while stimulating TC projections in acute brain slices containing theauditory thalamus (i.e., the ventral part of the medial geniculatenuclei [MGv]) and the ACx. TC projections in WT mice were nothaloperidol-sensitive at any age, whereas Df(16)1/+ TC projectionsbecame sensitive to haloperidol (1 μM) only at 3 months of age. In oldermice (4 months or older), TC EPSCs in the presence of haloperidolincreased approximately two fold compared to baseline TC EPSCs, whereasin younger mice (2 months or younger) TC EPSCs were not sensitive tohaloperidol. The Drd2 mRNA level was elevated in the auditory thalamusof 4-month-old (haloperidol-sensitive age) but not 2-month-old(haloperidol-insensitive age) Df(16)1/+ mice, and the input-outputrelation of TC synaptic transmission was deficient in 4-month-old butnot 2-month-old Df(16)1/+ mice. Consistent with the notion of aDgcr8-miRNA-Drd2 mechanism of TC deficiency in 22q11DS⁶, TC projectionsof Dgcr8^(+/−) mice but not WT littermates were sensitive to haloperidolonly after 3 months of age. Drd2 mRNA levels were also elevated in thethalamus of 4-month-old but not 2-month-old Dgcr8^(+/−) mice. Impairedglutamate release at TC projections causes an abnormal flow of acousticinformation to the ACx, resulting in a decreased acoustic-startleresponse and impaired prepulse inhibition (PPI) of acoustic startle⁶, ameasure of sensorimotor gating that is typically reduced inschizophrenic patients^(21,22). Consistent with the notion of a lateonset of schizophrenia symptoms, PPI deficits had a late onset, beingpresent in 4-month-old but not 2-month-old Dgcr8^(+/−) mice.

Basal synaptic transmission was compared in young (2-month-old) andmature (4-month-old) Df(16)1/+ mice, a murine model of 22q11DS⁴⁰ (FIG.1a ), and their wild-type (WT) littermates. Using whole-cellvoltage-clamp recordings, TC excitatory postsynaptic currents (EPSCs)were measured from the cortical layer (L) 3/4 pyramidal neurons, themain thalamorecipient neurons in the ACx⁷³, while stimulating TCprojections in acute brain slices containing the auditory thalamus(i.e., the ventral part of the medial geniculate nuclei [MGv]) and theACx (FIG. 1b ). The input-output relation between stimulation intensityand TC EPSC, a measure of basal synaptic transmission at TC projections,was deficient in older but not younger mutant mice (white circles)compared to WT (black circles) controls (FIG. 1 c, 1 d). Consistent withthe notion that the Drd2 elevation in thalamic-relay neurons reducesglutamatergic synaptic transmission at auditory TC projections inDf(16)1/+ mice³⁹, the Drd2 mRNA level was elevated in the MGv of olderbut not younger Df(16)1/+ mice (right gray bars compared to WT shown inthe left black bars) (FIG. 1e ).

Elevated Drd2 levels in older Df(16)1/+ mice mediate the abnormalsensitivity of mutant TC projections to antipsychotics; thus, the timecourse of this sensitivity was tested at different postnatal ages (1.5-7months). In brief, the thalamic radiation of WT mice was stimulated toevoke TC EPSCs with a rise slope of approximately 100 pA/ms. The effectof the antipsychotic agent haloperidol on TC EPSC 30 minutes after itsbath application was compared to the preapplication baseline TC EPSC(ΔH). Using ΔH as a measure of haloperidol sensitivity, it wasdetermined that TC projections in WT mice were not sensitive tohaloperidol at any age. However, Df(16)1/+ TC projections becamesensitive to haloperidol (1 μM) in an age-dependent manner. The ΔH wassignificantly higher in Df(16)1/+ mice (white circles) than in WTlittermates (black circles) but only beginning at 3 months of age (FIG.1f-1h ). In older mice, a similar intensity of thalamic stimulation(523±42 μA, n=31 in WT and 550±39 μA, n=30 in Df(16)/+ mice; p>0.05)applied to the thalamic radiation evoked substantially smaller TC EPSCsin Df(16)1/+ mice (white circles) compared to WT controls (blackcircles), and haloperidol rescued that deficit (FIG. 1f,1g ). Incontrast, TC projections in younger mutant mice (white circles) were notsensitive to haloperidol at similar stimulation intensities relative toWT mice (black circles) (527±61 μA, n=19 in WT and 568±50 μA, n=21 inDf(16)/+ mice; p>0.05) (FIG. 1h ).

Consistent with the notion that Dgcr8 underlies the TC deficiency in22q11DS³⁹, TC projections in Dgcr8^(+/−) mice (gray circles) but not WTmice (black circles) were sensitive to haloperidol at older than 3months of age, at similar stimulation intensities of the thalamicradiation (543±43 μA, n=36 in WT and 551±43 μA, n=37 in Dgcr8^(+/−)mice; p>0.05). TC projections in younger mice were not sensitive tohaloperidol (569±44 μA, n=16 in WT and 582±37 μA, n=24 in Dgcr8^(+/−)mice; p>0.05) (FIG. 1i-1k ). Drd2 mRNA levels were also elevated in thethalamus of only the older Dgcr8^(+/−) mice (shown in right gray barscompared to WT, shown in left black bars) (FIG. 11). Furthermore, thePPI, a measure of sensorimotor gating that is typically reduced inschizophrenic patients^(74,75), was deficient in older but not youngerDgcr8^(+/−) mice (shown in right gray bars compared to WT, shown in leftblack bars) (FIG. 1m,1n ). These data indicate that the pathogenicDgcr8-Drd2 mechanism of 22q11DS underlies the disruption of TC synaptictransmission but only later in life.

Example 3: miR-338-3p Mediates the Disruption of TC SynapticTransmission in 22q11DS

Because Dgcr8 is involved in miRNA processing⁷¹, the inventors sought toidentify the miRNA(s) mediating the Dgcr8-Drd2 mechanism of TCdeficiency. To this end, miRNA microarray analysis of the auditorythalamus of 2- and 4-month-old mice was performed (See Table 1, below).Among miRNAs that potentially target the Drd2 transcript (based onmiRWalk and Exiqon miRNA target-prediction algorithms providingpredicted seed-site sequences) in Drd2 3′ UTR, only only five miRNAs(miR-337-3p, miR-337-5p, miR-335-5p, miR-335-3p, and miR-338-3p) weredepleted in the auditory thalamus of Df(16)1/+ mice or Dgcr8^(+/−) mice(FIG. 2a-2d ). Because miR-185, which is not a Drd2-targeting miRNA, isencoded within the Df(16)1 microdeletion, its substantial depletion inDf(16)1/+ mice served as a positive control (FIG. 2a, 2b ). QRT-PCRanalysis verified that all five identified Drd2-targeting miRNAs weredepleted in Dgcr8^(+/−) mice (second lighter gray and fourth/last darkergray bars of each miRNA) compared to that in WT littermates (firstlightest gray and third black bars of each miRNA) at both ages (FIG. 7).Interestingly, the expression of miRNAs decreased with age, regardlessof genotype. The levels of identified miRNAs in older mice were lowerthan those in young WT or Dgcr8^(+/−) mice. However, because Dgcr8haploinsufficiency depleted these miRNAs at both ages, the age-dependentdecline in miRNA expression was exacerbated in mutants and reachedminimal values at 4 months in Dgcr8^(+/−) mice (fourth/last darker graybar of each miRNA) (FIG. 7). This age-dependent decline was exacerbatedin Dgcr8^(+/−) mutants and reached the minimal values in 4-month-oldDgcr8^(+/−) mice.

To identify which miRNA(s) targeting the Drd2 3′UTR (FIG. 2e ) regulatesthe Dgcr8-Drd2 mechanism of TC deficiency, a screen was performed basedon the abnormal sensitivity of TC projections to haloperidol. To thisend, miRNAs were overexpressed in excitatory thalamic neurons byinjecting adeno-associated viruses (AAVs) encoding miR-337-5p,miR-338-3p, miR-335-5p, miR-337-3p, or miR-335-3p under control ofexcitatory neuron-specific promoter CamKIIα into the MGv of 4-month-oldDf(16)1/+ and WT mice (FIG. 2f,2g ). Overexpression of individual miRNAsin Df(16)1/+ (right gray bars) mice not only replenished the depletedmiRNA levels but elevated them beyond those in WT mice (left black bars)(FIG. 8a-8e ). However, of the five miRNAs, only miR-338-3poverexpression rescued the abnormal haloperidol sensitivity in Df(16)1/+mice (FIG. 2h (right gray bars compared to WT shown in left black bars),8f-8k (gray lines compared to WT shown in black lines)). Overexpressionof miR-338-3p in the MGv of Df(16)1/+ mice decreased Drd2 mRNA levels inthe MGv by 47.6%±10.2% compared to the control virus (n=6 mice forAAV-GFP-miR-338-3p and 6 mice for AAV-GFP; p<0.01), confirming thatmiR-338-3p regulates Drd2 levels. These data suggested that miR-338-3pis the culprit miRNA, and its depletion results in the TC synapticabnormalities in 22q11DS mice.

The following data further suggested that miR-338-3p is the culpritmiRNA: miR-338-3p overexpression in the MGv substantially decreased Drd2levels in the MGv (FIG. 10a -10 c; left black bars represent WT; middledark gray bars represent miR-338^(+/−); right light gray bars representmiR-338^(−/−) in 10b, 10 c). The miR-338-3p was among other miRNAs(miR-335-3p and miR-335-5p) that have conserved seed sites in the mouseand human Drd2 3′ UTR (miRWalk algorithm)⁵⁸. Consistent with the notionthat only more abundant miRNAs effectively regulate the targetingtranscript⁵⁹, miR-338-3p appeared to be more crucial for Drd2 regulationin the auditory thalamus than did miR-337-5p, miR-335-5p, miR-337-3p, ormiR-335-3p. Indeed, miR-338-3p was enriched in the thalamus compared tothe other 4 miRNAs, and the levels of miR-337-3p, miR-335-5p,miR-337-3p, or miR-335-3p ranged between approximately 0% and 4% that ofmiR-338-3p (FIG. 2i ). Moreover, miR-338-3p was substantially enrichedin the thalamus compared to other tested brain regions (FIG. 2i ),suggesting that depletion of this Drd2-regulating miRNA in 22q11DSmainly affects thalamic function. Similarly, miR-338-3p was enriched inthe MGv compared to the ACx (Brodmann area 41) in postmortem tissuesamples from human subjects (n=6 for both thalamus and ACx, p<0.001)(FIG. 2j ). Moreover, miR-338-3p was significantly decreased in thethalamus of schizophrenic patients (right gray bars) compared to that inage- and sex-matched controls (left black bars) (n=6 for bothconditions, p<0.05) (FIG. 2j ). The inventors previously showed that theDrd2 protein level is elevated in the same set of MGv samples³⁹.

Example 4: miR-338-3p Depletion is Sufficient to Trigger Sensitivity ofTC Projections to Antipsychotics; miR-338 Depletion in the MGv ormiR-338-Knockout Recapitulates the Auditory TC Synaptic Abnormalities of22q11DS Mice

To test whether miR-338-3p depletion is sufficient to triggersensitivity of TC projections to antipsychotics, two strategies wereemployed: (i) a miR-338-3p sponge' was constructed by using a previouslydescribed strategy⁷⁸, and (ii) generation of miR-338-knockout (KO) mice(FIG. 4d ). The miR-338-3p sponge efficiency was verified in an in vitrosystem by using the luciferase assay. The sponge with 12 seed sites wassufficient to almost completely and specifically deplete miR-338-3plevels according to this assay (FIG. 9). On the basis of these data,AAVs were constructed expressing either the miR-338-3p sponge or ascrambled control vector under the control of the CamKIiα promoter (FIG.4a ). The AAV expressing the miR-338-3p sponge that was injected intothe MGv of WT mice was sufficient to increase Drd2 mRNA (FIG. 4b ) andrender the TC projections sensitive to haloperidol (FIG. 4c ). Theexcitatory neurons in the MGv of WT mice were infected with AAVsexpressing either the miR-338-3p sponge or a scrambled control vectorunder the control of the CamKIla promoter (FIG. 4a ). The miR-338-3psponge (right gray bar) increased Drd2 mRNA in the auditory thalamusrelative to scrambled control (left black bar) (FIG. 4b ). ThemiR-338-3p sponge (gray) also rendered TC projections in WT miceabnormally sensitive to haloperidol, whereas the scrambled control(black) did not (FIG. 4c ). These experiments indicate that depletion ofmiR-338-3p is necessary and sufficient to increase Drd2 expression inthe thalamic-relay neurons and render TC projections to the ACxsensitive to antipsychotics.

Because miR-338-3p appears to be a major miRNA controlling Drd2expression in the auditory thalamus, a mutant mouse lacking miR-338(miR-338 KO mice) was generated (FIG. 4d ). The miR-338 is an intragenicmiRNA whose genomic locus is inside the seventh intron of the Aatk(apoptosis-associated tyrosine kinase) gene. However, unlike miR-338-3p,Aatk expression in the MGv was not affected by age (FIG. 10a ) ormiR-338 deletion (FIG. 10b ; left black bar corresponding to WT mice,middle dark gray bar corresponding to miR-338^(+/−) mice; right lightgray bar corresponding to miR-338^(−/−) mice). The miR-338 KO micelacked miR-338-3p, miR-338-5p, and miR-3065 (both -3p and -5p species)(FIG. 10c ), whose genomic locus overlaps with miR-338. HowevermiR-338-5p, miR-3065-3p, and miR-3065-5p were not Drd2-targeting miRNAs,as predicted by the microRNA target-prediction algorithms, and theirexpression level in the auditory thalamus were 0% and 2.5% that ofmiR-338-3p (FIG. 10 c; left black bar corresponding to WT mice, middledark gray bar corresponding to miR-338^(+/−) mice; right light gray barcorresponding to miR-338^(−/−) mice). The miR-338^(+/−) or miR-338^(−/−)mice developed normally without any gross morphological abnormalities(FIG. 10d ,10 e; left black bar corresponding to WT mice, middle darkgray bar corresponding to miR-338^(+/−) mice; right light gray barcorresponding to miR-338^(−/−) mice in 10e). Their Drd2 levels wereinversely correlated with miR-338-3p levels in the auditory thalamus(FIG. 4e ; left black bar corresponding to WT mice, middle dark gray barcorresponding to miR-338^(+/−) mice; right light gray bar correspondingto miR-338^(−/−) mice), further indicating that miR-338-3p is thecritical regulator of Drd2 expression in the auditory thalamus.

Because miR-338-3p is depleted but not eliminated in Df(16)1/+ mice, TCsynaptic properties were tested in 4-month-old miR-338^(+/−) mice. Likethat in Df(16)1/+ mice⁵, synaptic transmission at TC projections wassubstantially disrupted in miR-338^(+/−) mice. The input-outputfunction, which were tested by electrical stimulation of TC projections,showed a significant (p<0.01) decrease in TC EPSCs in miR-338^(+/−) mice(gray or white circles) compared to that in WT mice (black circles)(FIG. 4f,4g ). This disruption was specific to TC projections. Theinput-output function tested by electrical stimulation ofcorticocortical (CC) projections in the same slices did not differbetween miR-338^(+/−) (white circles) and WT mice (black circles) (FIG.4h ). The PPR of two consecutive electrically evoked EPSCs wassubstantially altered in TC but not CC projections of miR-338^(+/−) mice(gray or white circles) compared to that in WT (black circles) controls(FIG. 4i,4j ). Specifically, FIG. 4i shows an increase in the PPR of twoconsecutive electrically evoked EPSCs of the miR-338^(+/−) mice (topgray line, n=22) relative to WT mice (bottom black line, n=26). Incontrast, the NMDAR/AMPAR ratio (a measure of the postsynaptic function)was normal in both TC and CC projections of miR-338^(p+/−) mice (rightgray bars compared to WT, shown in left black bars) (FIG. 4k,4l ).Because electrical stimulation of the thalamic radiation may affectcircuits other than TC projections⁷⁹, TC projections were activated moreselectively using the optogenetic approach. To that end, AAVs expressingChR2 under the control of CamKIIα were injected into the MGv ofmiR-338^(+/−) and WT littermates. TC projections were then activated byusing 473-nm light pulses (FIG. 4m ). The input-output relations and PPR(but not the NMDAR/AMPAR ratio of optically evoked EPSCs weresubstantially decreased in 4-month-old miR-338^(+/−) mice (white circlesor gray bars) compared to that in WT littermates (black circles or blackbars) (FIG. 4n -4 p; data from WT mice shown in left black bar andmiR-338^(+/−) mice shown in right gray bar in p), which recapitulatedthe TC disruption in 22q11DS mouse models.

Example 5:miR-338 Haploinsufficiency Disrupts TC Projections byDecreasing the Probability of Glutamate Release from ThalamicProjections

The inventors have previously shown that the TC disruption of synapticplasticity in 22q11DS mouse models was due to defective presynapticfunction, which was in turn was caused by reduced probability ofglutamate release from thalamic projections³⁹. Abnormalities in theinput-output relation and PPR at TC projections of miR-338^(+/−) micealso suggested a deficit in presynaptic function at TC glutamatergicsynapses. To understand the nature of this deficit, two-photon calciumimaging was performed in dendritic spines, which are the inputs ofthalamic projections onto thalamorecipient neurons in the ACx. L3/4pyramidal neurons were loaded with the calcium indicator Fluo-5F andcytoplasmic dye Alexa 594 (FIG. 5a ) and dendritic spines that respondedto electrical stimulation of the thalamic radiation were identified(FIG. 5b ). This method enabled measurement of three factors that maycontribute to the TC disruption: the distribution of synaptic inputs ondendritic trees of postsynaptic neurons, the amplitudes of calciumtransients, and the probability of calcium transients at individualdendritic spines (a proxy for the probability of neurotransmitterrelease measured at a single synaptic input)^(5, 80, 81). Thedistribution of active TC inputs on dendritic trees and the peakamplitudes of postsynaptic calcium transients in miR-338^(+/−) mice(gray bars or white circles) were comparable to that in WT mice (black)(FIG. 5c-5e ), suggesting that TC development, pathfinding, synaptictargeting of cortical neurons by TC projections, and postsynapticglutamatergic receptor function were not compromised in miR-338^(+/−)mice. However, the probability of calcium transients in dendritic spinesof thalamorecipient neurons in response to a low-frequency (0.1 Hz)train of stimuli was deficient in miR-338^(+/−) mice (FIG. 5f ). Thisresult indicates that the depletion of miR-338 decreased the probabilityof glutamate release at TC projections, which underlies the TCdisruption in 22q11DS. Data from 27 WT mice shown in left black bars anddata from 32 miR-338^(+/−) mice shown in right gray bars in FIG. 5d -5f.

Exampld 6: miR-338 Depletion Eliminates the Age Dependency of TCDisruption and PPI

The deletion of miR-338 was sufficient to upregulate Drd2 in thethalamus, which suggested that depletion of only this miRNA underliesthe abnormal sensitivity of TC projections in 22q11DS to antipsychotics.To test this hypothesis, the sensitivity of TC projections inmiR-338^(+/−) mice and WT mice was compared. First, it was determinedthat TC projections of miR-338^(+/−) (white) but not WT (black) micewere sensitive to the Drd2-specific antagonist L-741,626 (20 nM) (FIG.6a ). In miR-338^(+/−) mice, TC EPSCs substantially increased inresponse to L-741,626, but that increase was not further elevated byhaloperidol. Haloperidol alone increased TC EPSCs in 4-month-oldmiR-338^(+/−) (white) mice (but not in WT (black) mice) to magnitudessimilar to those observed in Df(16)1/+ (white) or Dgcr8^(+/−) (gray)mice, suggesting that haloperidol's effect in mutant TC projections wasmediated by elevated expression of Drd2 receptors (FIGS. 6b, 11a-11c ).Similarly, other antipsychotics (i.e., clozapine and olanzapine)increased TC EPSCs to similar magnitudes in miR-338^(+/−) mice (white)but not WT (black) mice (FIG. 12a-12b ).

Unlike Df(16)1/+ or Dgcr8^(+/−) mice, miR-338^(+/−) mice (white) becamesensitive to haloperidol in an age-independent manner (FIGS. 6b, 11a-11c). Furthermore, in young (2-month-old) WT mice, TC projections becamesensitive to haloperidol when the miR-338-3p sponge (light gray) wasexpressed in the MGv (FIG. 13a,13b ), indicating that depletion ofmiR-338-3p in the MGv is sufficient for sensitivity to antipsychotics.TC sensitivity to haloperidol in 2-month-old miR-338^(+/−) (white) micewas eliminated by expression of small inhibitory RNA (siRNA) againstDrd2 (but not a control siRNA) in the MGv relative to WT (black) mice(FIG. 6c,6d ), which further indicated that miR-338-3p is sufficient toregulate Drd2 in the thalamus regardless of age. Similarly,miR-338^(+/−) mice (right gray bars) were deficient in PPI compared tothat in WT controls (left black bars), and this deficit was observed atall tested time points (1.5, 2, and 4 months) (FIG. 6e-6g ). The defectin PPI was not caused by peripheral hearing defects because the acousticbrainstem-response testing showed no differences between miR-338^(+/−)(white) and WT (black) mice at these ages (FIG. 14a-14c ).

Like the TC projections in Df(16)1/+ mice and Dgcr8^(+/−) mice, those inmiR-338^(+/−) (white) mice showed abnormal sensitivity to haloperidolrelative to WT (black) mice (FIG. 6a ,6 b; FIG. 11), or otherantipsychotics (clozapine and olanzapine) (FIG. 12a -12 b; data from WTmice shown in black and data from miR-338^(+/−) mice shown in white),and that sensitivity was mediated by Drd2 receptors. Indeed, TC EPSCswere substantially increased in the presence of the specific Drd2inhibitor L-741,626 (20 nM), and this increase was not further elevatedby haloperidol (FIG. 6a ). Haloperidol alone also increased TC EPSCs in4-month-old miR-338^(+/−) (white) mice relative to WT (black) mice (FIG.11a ) to magnitudes similar to that observed in Df(16)1/+ or Dgcr8^(+/−)mice (p>0.05). However, in contrast to Df(16)1/+ or Dgcr8^(+/−) mice,miR-338^(+/−) mice became sensitive to haloperidol in an age-independentmanner (FIG. 6b ; FIG. 11a -11 c; in these figures, data from WT miceare represented in black while data from miR-338^(+/−) mice arerepresented in white). Furthermore, TC projections became sensitive tohaloperidol in 2-month-old WT mice when the miR-338-3p sponge (lightgray) but not the scrambled control (black) was expressed in the MGv(FIG. 13a,13b ). TC sensitivity to haloperidol in 2-month-oldmiR-338^(+/−) (white) mice was eliminated by local MGv expression shorthairpin RNA (shRNA) against Drd2 (but not control shRNA) relative to WT(black) mice (FIG. 6c, 6d ). Similarly, miR-338^(+/−) mice (right graybars) were deficient in PPI compared to WT controls (left black bars),and this deficit was observed at early time points (1.5, 2 and 4 months)(FIG. 6e-6h ). Acoustic brainstem response (ABR) showed no hearingdeficits in miR-338^(+/−) (white) mice at these ages relative to WT(black) mice (FIG. 14).

Example 7: Prepulse Inhibition (PPI) in miR-338 Mice

Mouse behavioral tests. PPI experiments were performed as previouslydescribed⁶. Briefly, each day before testing, the mice were allowed a 1hour habituation period in the testing room, after being transportedfrom the animal-housing room. Before experiments were initiated, themice were allowed to acclimate to the Plexiglas restraint chamber (6cm×6 cm×4.8 cm) for 20 min. The mice had a 5-min acclimation period to a65-dB background white noise, which played throughout the session. ForPPI experiments, three acoustic startles (8 kHz, 120 dB, 40 ms) weredelivered separated by a 15-s intertrial interval. The testing sessionconsisted of 39 trials of 5 trial types: pulse-alone in which thestartle pulse was presented, the combination of a 40-ms prepulse (74 dB,82 dB, or 90 dB) in WT and Dgcr8^(+/−) littermates and (70 dB, 80 dB, 90dB) in WT and miR-338^(+/−) littermates and preceding the startle pulseby 100 ms, and no stimuli. Trials were separated by 15 s and presentedin a pseudo-random order. PPI was calculated as follows:100×(pulse-alone response−prepulse+pulse response)/pulse-alone response.

miR-338^(+/−) mice (right gray bars) were deficient in PPI compared toWT controls (left gray bars), and this deficit was observed at earlytime points (1.5 and 2 months) (FIG. 6e,6f ). Acoustic brainstemresponse (ABR) showed no hearing deficits in miR-338^(+/−) (white) miceat these ages relative to WT (black) mice (FIG. 14a-14c ).

Example 8: Nasal Delivery of miR-338-3p Mimic

The miR-338-3p mimic (double-stranded synthetic RNA, 22nt in length)UCCAGCAUCAGUGAUUUUGUUG (SEQ ID NO: 1; the same sequence as the sequenceof the mature miR-338-3p) was purchased from GE Dharmacon (Cat#c-310547-07-0020). The mimic was resuspended in nuclease-free water tomake a stock solution at 1.41 μg/μl. The working solution was 0.25 μg/μlin PBS (PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO₄, pHof 7.4). 10 μl of working solution was intranasally injected to eachnostril of a mouse. miR-338-3p levels were then measured at differenttime points in different brain regions using quantitative RT-PCR(qRT-PCR). It was found that the miR-338-3p mimic accumulates in thethalamus but not hippocampus or cortex or striatum after 10 minutesafter delivery.

CONCLUSIONS

These data indicate that miR-338-3p is the miRNA that mediates theDgcr8-miRNA-Drd2 pathogenic pathway in TC projections to the ACx. Dgcr8haploinsufficiency in 22q11DS depletes this thalamus-enriched miRNA,which leads to Drd2 upregulation in thalamic-relay neurons. Severalstudies have indicated that drug-naive schizophrenic patients haveelevated levels of DRD2s in their brains^(25,28). Drd2 upregulationleads to deficits in TC synaptic transmission and acoustic startle andalso renders TC projections sensitive to antipsychotics. Antipsychoticsthat effectively treat only positive symptoms but not cognitive ornegative symptoms of the disease²⁹, eliminate synaptic deficits at TCprojections and acoustic-startle deficiency in 22q11DS mice⁶. AlthoughmiR-338-3p is depleted from the auditory thalamus at all ages in 22q11DSmice, it declines even more with age and reaches a low enough level inyoung adult 22q11DS mice to trigger Drd2 upregulation and TC andacoustic-startle deficiencies. This suggests that miR-338-3p is the keyregulator of the late onset of positive symptoms of 22q11DS-associatedschizophrenia. Antipsychotics alleviate positive symptoms ofschizophrenia through systemic inhibition of DRD2, which is accompaniedby multiple and sometimes devastating side effects^(2,30). The dataprovided herein suggest that replenishing miR-338-3p in the thalamus ofschizophrenic patients could be a promising and more tolerabletherapeutic approach to treating schizophrenia-associated psychosis.

The recent identification of disrupted glutamatergic synaptictransmission at thalamic inputs to the ACx in 22q11DS mice³⁹ suggeststhat TC disruption could be a pathogenic mechanism that mediates thesusceptibility to positive psychotic symptoms in 22q11DS-relatedschizophrenia for the following reasons: 1) TC disruption in 22q11DSmice is rescued by antipsychotic medications that are Drd2 antagonistsand effectively treat predominantly psychotic symptoms but not cognitiveor negative symptoms of schizophrenia⁸²⁻⁸⁴. This disruption was specificto auditory TC projections and was not observed at other glutamatergicprojections (i.e., hippocampal, corticocortical, or corticofugalprojections) that may be involved in cognitive, social, or motivationaltasks. 2) TC disruption in 22q11DS mice is caused by abnormal elevationof Drd2 mRNA and Drd2 protein levels in the TC neurons in the thalamus,a brain region previously linked to psychotic symptoms ofschizophrenia⁸⁵⁻⁸⁸. The increase in dopamine signaling in the thalamuswas also described in schizophrenic patients⁸⁹, and studies haveindicated that drug-naive schizophrenic patients have elevated levels ofDRD2s in other brain regions^(90,91). Furthermore, theoretical andempirical studies have proposed that deficient connectivity and abnormalpatterns of activity in TC projections contribute to the pathogenesis ofthe disease⁹²⁻¹⁰¹. Moreover, a local ischemic infarction that disruptsauditory TC projections in a nonpsychotic patient can cause auditoryhallucinations¹⁰². 3) Sensitivity to antipsychotics is observed in theauditory but not the visual or somatosensory TC projections of 22q11DSmice, which is consistent with clinical observations of thesubstantially higher prevalence of auditory hallucinations, comparedwith that of hallucinations in other sensory modalities, inschizophrenia^(63, 103-105). Neuroimaging and electrophysiologicalstudies in schizophrenia patients have shown abnormal activation of theauditory thalamus and ACx during auditoryhallucinations^(35-37, 57, 106-112); 4) Drd2 elevation only in theauditory thalamus of 22q11DS mice was sufficient to reduce the PPI ofthe acoustic startle response⁵, the behavioral endophenotypecharacteristic of patients with one of several psychiatric diseases,including 22q11DS and schizophrenia^(75, 113).

Here it was shown that the disruption of synaptic transmission atauditory TC projections recapitulates another prominent feature ofpsychotic symptoms. The TC disruption in 22q11DS mice becomes evidentonly in late juvenile or adult mice, which mirrors clinicalmanifestations of psychosis in patients with 22q11DS or schizophreniaduring late adolescence or early adulthood, typically between the agesof 16 and 30 years^(61, 114). This age-dependent TC decrease in synapticfunction is evident in Df(16)1/+ mice, which carry a largemicrodeletion, and in Dgcr8^(+/−) mice, further strengthening the casethat Dgcr8 is the culprit gene, and its haploinsufficiency underliesauditory abnormalities in 22q11DS.

Previous work established that the deletion of one copy of Dgcr8 leadsto the elevation of Drd2 in the auditory thalamus³⁹. Because Dgcr8 ispart of the miRNA-processing machinery, it was hypothesized that aDgcr8-miRNA-Drd2 mechanism underlies the disruption of TC synaptictransmission. Here miR-338-3p was identified as the mediator of thismechanism. It is also shown herein that miR-338-3p negatively regulatedthe level of Drd2 in the thalamus. Replenishing miR-338-3p in thethalamus eliminated deficient TC synaptic transmission and abnormalantipsychotic sensitivity of TC projections in 22q11DS mice, and thedeletion or auditory thalamus-specific knockdown of miR-338-3p mimickedTC disruption of synaptic transmission and antipsychotic sensitivity inWT mice. Depletion of miR-338-3p is therefore necessary and sufficientto upregulate Drd2, which in turn, reduces glutamate release fromthalamic projections, reduces TC synaptic transmission, and renders TCprojections sensitive to antipsychotics.

One copy of Dgcr8 is deleted in 22q11DS, in all cells at all ages, so itis unclear why synaptic disruption occurs in projections emanating onlyfrom the thalamus and only later in life. The regional specificity mostlikely arises from the fact that miR-338-3p is substantially enriched inthe auditory thalamus compared to other tested brain regions, such asthe cortex or hippocampus. Explaining why miR-338-3p isthalamus-enriched will require further investigation. It was alsodetermined that the expression of miR-338-3p is regulated in anage-dependent manner. Although miR-338-3p is depleted in the auditorythalamus in 22q11DS mice at all ages, compared to WT mice, it declinesfurther with age in both 22q11DS and WT mice. Therefore, miR-338-3pexpression may be controlled by a combination of Dgcr8- andage-dependent mechanisms. Although it may be possible that Dgcr8haploinsufficiency reduced the levels of miRNAs, the mechanism ofage-dependent miRNA decline is unknown. In the context of Drd2regulation, a minimal threshold of miR-338-3p expression probablytriggers the overexpression of Drd2. In WT mice, miR-338-3p declinesduring the first few months of life, but it may not reach thatthreshold. However, in 22q11DS mice, Dgcr8 haploinsufficiency andage-dependent decline in miRNA production drives the miR-338-3p levelbelow this threshold, triggers the elevation of Drd2 in the thalamus,and causes TC synaptic and behavioral deficiencies.

The data provided herein implicate thalamus-enriched miR-338-3p as thekey mediator of disruption of synaptic transmission at TC projectionsand the regulator of the late onset of auditory symptoms of 22q11DS.These data also suggest that replenishment of miR-338-3p in the thalamuscould be a more tolerable therapeutic approach for positive symptoms.Current therapy relies upon antipsychotics to alleviate psychosis inschizophrenic patients through systemic inhibition of DRD2, which isaccompanied by multiple, and sometimes devastating, sideeffects^(62, 115). Given that the seed sites of miR-338-3p are conservedbetween mice and humans and miR-338-3p is depleted in the thalamus ofmouse models of 22q11DS and schizophrenic patients, this strategy ispotentially applicable to patients. Thus, these results suggest thatmiR-338-3p is a potential therapeutic target for treating positivesymptoms of 22q11DS and related cases of schizophrenia.

TABLE 1 Altered miRNA levels in the auditory thalamus of 2- and4-month-old Df(16)1/+ and Dgcr8^(+/−) mice compared to respective WTlittermates 2 months 4 months Df(16)1/+, Df(16)1/+, Dgcr8^(+/−),Dgcr8^(+/−), Df(16)1/+, Df(16)1/+, Dgcr8^(+/−), Dgcr8^(+/−), miRNA ID*log₂(FC) −lgP log₂(FC) −lgP log₂(FC) −lgP log₂(FC) −lgP miR-185-5p−1.64858 7.33282 −0.38928 4.36128 −1.78536 13.30000 −0.50611 5.60000miR-340-5p −0.45282 4.25303 −0.41144 4.74020 −0.52815 7.67000 −0.432016.34000 miR-379-5p −0.52811 2.34417 −0.57980 6.79574 −0.49632 5.72000−0.56681 4.75000 miR-337-5p −0.43430 3.43963 −0.44315 3.97221 −0.492904.79000 −0.38033 5.28000 miR-874-3p −0.22839 1.35784 −0.36327 6.04564−0.42984 3.50689 −0.32809 3.01403 miR-151-5p −0.29580 2.62959 −0.159351.22879 −0.42306 5.32000 −0.42340 3.84858 miR-490-5p −0.29997 3.33885−0.30096 4.76328 −0.40860 2.66448 −0.28810 2.79992 miR-337-3p −0.725943.88408 −0.53059 4.67241 −0.40453 3.51575 −0.56095 5.32000 miR-194-5p−0.09643 0.49292 −0.19463 2.31803 −0.39231 4.47000 −0.14971 1.17787miR-412-3p −0.26047 1.19135 −0.02458 0.15986 −0.38210 2.74342 −0.499775.77000 miR-186-5p −0.30806 3.30560 −0.35820 4.20053 −0.36877 4.54000−0.29587 3.81030 miR-376c-3p −0.44510 2.65559 −0.37691 3.55672 −0.365652.38980 −0.25579 3.25004 miR-491-3p −0.02866 0.17304 −0.02291 0.16779−0.36420 2.31817 −0.28696 3.82584 miR-873a-5p −0.39810 2.22932 −0.465132.86648 −0.35986 3.16223 −0.44403 3.82988 miR-378a-3p −0.02597 0.03877−0.29050 2.03499 −0.35799 1.17895 −0.12968 0.58938 miR-674-3p −0.406652.47569 −0.26268 3.18660 −0.34936 3.18669 −0.38926 4.33000 miR-411-5p−0.52946 5.45327 −0.48809 5.03545 −0.34928 2.50056 −0.51329 5.49000miR-99b-5p 0.04461 0.17949 −0.16827 2.00753 −0.34897 3.38707 −0.148511.83458 miR-361-5p −0.26047 2.24137 −0.18554 2.30066 −0.34870 4.91000−0.24978 3.89852 miR-341-3p −0.24355 1.77885 −0.24290 2.95254 −0.335626.25000 −0.30452 4.68000 miR-338-3p −0.19175 1.36799 −0.10823 0.99242−0.33405 3.04168 −0.21677 2.55006 miR-674-5p −0.17303 1.19746 −0.176432.94184 −0.33270 4.40000 −0.16778 3.25601 miR-488-5p −0.00195 0.00805−0.02020 0.21260 −0.32913 2.05711 −0.25677 2.89363 miR-323-3p −0.370961.82575 −0.37050 4.17116 −0.32876 2.97886 −0.36882 3.56822 miR-218-5p−0.17671 0.99892 −0.11072 1.71566 −0.31911 4.28000 −0.30454 2.68477miR-27b-3p −0.04949 0.17414 −0.08396 0.44242 −0.30721 2.57126 −0.080520.23717 miR-539-3p 0.14778 0.70416 0.00498 0.03638 −0.29926 4.58000−0.35779 3.54966 miR-25-3p −0.22723 1.88239 −0.27461 2.63331 −0.297553.10974 −0.32216 2.98604 miR-329-3p −0.56459 2.21457 −0.41486 3.46643−0.27725 1.71625 −0.55649 4.08000 miR-148b-3p −0.34932 2.27549 −0.207952.63388 −0.27550 1.74510 −0.33757 5.00000 miR-134-5p −0.09379 0.47169−0.15267 2.72667 −0.27550 3.62664 −0.11857 1.85538 miR-219-5p −0.247070.70032 0.13979 0.47249 −0.27438 2.96830 −0.03437 0.15228 miR-130b-3p−0.16519 1.40819 −0.29858 3.87589 −0.27129 5.18000 −0.33165 6.06000miR-331-3p −0.27610 2.75741 −0.25452 2.60266 −0.26460 3.51172 −0.420866.19000 miR-299b-5p −0.64210 2.95443 −0.40837 3.10344 −0.26125 2.45913−0.44087 5.20000 miR-409-5p −0.34006 2.92694 −0.39965 2.87777 −0.258151.83035 −0.45668 6.03000 miR-374c-5p −0.40264 1.96791 −0.42464 3.88306−0.25437 1.92815 −0.41823 2.04837 miR-362-3p −0.40304 1.70048 −0.093420.44505 −0.24979 3.65603 −0.22090 1.98880 miR-582-5p −0.30107 2.91016−0.35764 4.56001 −0.24543 2.26635 −0.28238 3.74804 miR-21a-5p −0.225311.42508 0.00924 0.06228 −0.23955 2.13251 −0.11702 1.30260 miR-378a-5p−0.09892 0.28496 −0.18501 2.43253 −0.23859 1.35479 −0.19665 1.25410miR-410-3p −0.04583 0.26667 −0.14896 0.98136 −0.23242 1.70776 −0.432185.01000 miR-382-5p −0.21794 1.92872 −0.16890 1.89499 −0.23210 1.18307−0.35547 4.17000 miR-342-3p −0.30374 1.52055 −0.46348 4.30075 −0.225591.61458 −0.23434 2.68110 miR-532-5p −0.33157 5.00883 −0.28830 2.44847−0.22199 2.52259 −0.29624 2.28342 miR-98-5p −0.36761 1.74004 −0.188482.26707 −0.21721 1.25601 −0.21391 1.76160 miR-3072-3p −0.21737 2.06707−0.14584 1.67723 −0.21292 2.69576 −0.19259 3.33974 miR-192-5p −0.322672.33187 −0.08968 0.89149 −0.21239 2.14488 −0.24133 2.15092 miR-874-5p−0.18357 0.79526 −0.09993 1.07675 −0.20736 2.41490 −0.17490 3.89565miR-376b-5p −0.47709 2.32419 −0.32841 2.75823 −0.20095 0.91922 −0.244482.36934 miR-350-3p −0.50643 2.16582 −0.17792 1.73452 −0.18231 1.24276−0.45051 3.77820 miR-429-3p 0.02348 0.14990 −0.20844 1.35941 −0.176001.71161 −0.36166 4.45000 miR-22-3p −0.17417 0.88726 −0.29421 2.71705−0.17220 1.29505 −0.24582 1.44913 miR-770-3p −0.20164 0.92219 −0.305813.22754 −0.16802 0.62078 −0.11425 0.67675 miR-542-3p −0.17027 0.85234−0.04392 0.30919 −0.16405 1.21733 −0.35518 4.01000 miR-6540-5p −0.330392.00301 −0.23601 4.26359 −0.16165 1.32809 −0.17128 4.92000 miR-340-3p−0.58892 1.61555 −0.36654 3.89266 −0.15710 0.69413 −0.41571 4.87000miR-377-3p −0.33963 2.23645 −0.29085 1.42202 −0.14901 0.55745 −0.367754.75000 miR-540-5p −0.21736 2.59828 −0.29546 4.01680 −0.14786 1.95629−0.13382 2.68864 miR-325-3p −0.29095 3.39477 −0.28231 3.63211 −0.139841.73710 −0.32907 4.37000 miR-598-3p −0.52025 1.88248 −0.20415 1.18458−0.13708 0.49131 −0.27125 2.69229 miR-30e-5p −0.19170 1.94879 −0.328485.22315 −0.13560 0.56633 −0.18913 4.03000 miR-380-3p −0.51339 2.83322−0.31588 2.22996 −0.13458 0.53658 −0.33725 3.23072 miR-872-5p −0.308662.73200 −0.27909 2.60025 −0.13349 0.70013 −0.28532 2.58680 miR-590-3p0.15738 0.75091 −0.02917 0.18579 −0.12873 2.21882 −0.25609 3.35299miR-672-5p −0.20029 2.88575 −0.28012 2.12609 −0.11825 1.11673 −0.228812.48098 miR-369-3p −0.39108 2.24233 −0.38498 2.84318 −0.10938 0.91829−0.15479 0.64601 miR-7a-1-3p −0.32283 1.46454 −0.09042 0.60102 −0.103420.51267 −0.19373 2.15481 miR-154-3p −0.49966 2.01114 −0.22055 2.21980−0.09424 0.48481 −0.19292 3.36326 miR-379-3p −0.21497 2.16166 −0.288795.91856 −0.08758 0.86134 −0.21315 4.12000 miR-708-5p −0.13213 1.19633−0.27051 3.62166 −0.08592 1.09677 −0.09455 1.94207 miR-541-5p −0.386452.46292 −0.34754 4.12669 −0.07760 0.94494 −0.08278 1.30955 miR-18950.27858 2.18624 0.31776 1.59982 −0.07710 0.36636 0.20538 1.38773miR-22-5p −0.24476 1.30886 −0.16246 1.64132 −0.07575 0.45060 −0.173622.12231 miR-134-3p −0.23693 0.78101 −0.08546 0.57698 −0.06132 0.65616−0.14752 2.10755 miR-382-3p −0.44909 2.08070 −0.15171 1.11672 −0.059200.18686 −0.31600 3.42649 miR-425-5p −0.30094 3.25247 −0.27174 3.46697−0.05856 0.97021 −0.09711 1.62695 miR-411-3p −0.49774 3.28048 −0.532287.26456 −0.05174 0.98963 −0.12142 1.79691 miR-346-5p −0.23181 3.15968−0.19143 2.68474 −0.04945 0.49158 −0.19230 2.64636 miR-153-5p −0.158741.94310 −0.30419 3.44445 −0.04745 0.27636 −0.15579 3.03991 miR-190a-5p−0.29029 2.33972 −0.31362 2.85912 −0.04481 0.26848 −0.16720 1.34855miR-544-5p −0.22132 2.62262 −0.21683 2.93229 −0.03407 0.34004 0.062471.25639 miR-3471 0.24013 2.35467 0.17376 0.93282 −0.02954 0.197980.04906 0.25493 miR-149-3p 0.09333 1.02888 0.24249 2.60388 −0.026590.10003 0.15764 2.03132 miR-34a-5p −0.07092 0.19498 0.50004 2.28343−0.02252 0.07289 0.09950 0.28604 miR-496a-3p −0.34340 1.91396 −0.247993.73538 −0.02130 0.16834 0.03649 0.39580 miR-409-3p −0.34725 2.58636−0.49010 4.49122 −0.01837 0.14513 −0.00318 0.02532 miR-335-3p −0.297982.75084 −0.20560 3.02902 −0.01683 0.12972 −0.13894 1.12976 miR-490-3p−0.30625 2.73738 −0.28681 2.82531 −0.01141 0.06426 0.01475 0.22898miR-383-3p −0.21411 2.19625 −0.10800 0.90341 −0.00413 0.03477 −0.066330.58312 miR-30d-5p 0.33931 2.55178 0.17116 2.38771 −0.00367 0.022510.11727 1.93914 miR-488-3p −0.20675 2.30971 −0.33473 2.45405 −0.003130.02364 0.03016 0.18918 miR-5130 0.20246 2.67639 −0.00745 0.027730.00103 0.00609 0.03125 0.37387 miR-30a-5p 0.21787 1.36947 0.135903.77359 0.00838 0.03681 0.14345 2.58741 miR-532-3p −0.20336 1.72588−0.18918 2.24134 0.01114 0.07689 −0.04412 0.37793 miR-484 0.255071.73495 0.20172 2.80090 0.01339 0.06238 0.20716 1.28962 miR-320-3p0.18748 1.13071 0.20191 2.59032 0.03484 0.34162 0.13569 2.03130 miR-19490.13067 0.60288 0.24027 3.36284 0.03821 0.30417 0.20394 1.98905miR-125a-3p 0.29640 1.90275 0.10266 0.68995 0.03930 0.29418 0.280822.05517 miR-5119 0.17465 1.32435 0.26507 2.11748 0.04429 0.50059 0.078771.09644 miR-30b-3p 0.20526 2.30825 −0.00929 0.07818 0.04585 0.551150.02587 0.22431 miR-384-3p −0.21385 2.65583 −0.14759 1.48268 0.050880.38527 −0.03325 0.25394 miR-466h-3p 0.37225 2.17701 0.18741 0.692640.05243 0.21414 0.23517 1.46279 miR-30a-3p 0.20022 1.36951 0.144732.40011 0.05288 0.47115 0.09631 1.42419 miR-291b-3p 0.21574 0.855590.01851 0.09757 0.05999 0.51289 0.11816 2.21480 miR-1186b 0.242011.26108 0.24245 1.18070 0.07062 0.50990 0.24937 2.36667 miR-669n−0.03443 0.19165 0.00754 0.03275 0.07976 0.67927 0.24220 2.14081miR-3082-5p 0.13470 0.25902 0.12509 0.30716 0.08752 0.56557 0.287662.68193 miR-582-3p −0.36581 3.16016 −0.28702 2.84446 0.09241 0.342830.31655 1.92822 miR-127-3p 0.31162 0.92037 0.22463 1.39840 0.127600.45312 0.24279 2.46742 miR-712-5p 0.19497 0.80946 0.27763 2.870370.13836 0.97133 0.13884 1.10864 miR-200a-3p 0.10958 0.54329 −0.206102.17228 0.14003 0.80942 −0.04499 0.47935 miR-5117-5p −0.03307 0.09115−0.00820 0.04658 0.15792 2.05862 0.20996 1.07294 miR-664-3p 0.191791.06561 0.20097 3.46731 0.16058 1.14929 0.23420 2.64985 miR-138-1-3p0.25216 3.41325 0.26724 2.10524 0.16076 1.43355 0.16703 1.60822 miR-7050.26694 1.80529 0.34896 2.29776 0.17633 1.54490 0.18320 1.05974miR-1843a-5p 0.10436 0.49439 0.11951 0.86070 0.17902 1.43543 0.282833.79383 miR-3095-3p 0.74549 2.33919 0.41334 1.36465 0.18069 1.123510.30474 1.03585 miR-3096b-3p 0.24325 3.51385 0.19245 4.17219 0.186433.05684 0.17585 2.29537 miR-5113 0.76604 3.36870 0.38103 1.67553 0.203101.45130 0.20785 2.30914 miR-677-3p 0.21203 1.05257 0.21105 2.548720.21137 2.38834 0.29067 2.97121 miR-335-5p −0.42834 3.19848 −0.185030.86468 0.22218 1.04593 −0.22129 2.07806 miR-1839-3p 0.12067 1.970010.18008 2.61633 0.22866 2.69554 0.12984 1.39494 miR-6402 −0.021510.04380 0.24261 0.49376 0.25885 1.56379 0.25769 2.72355 miR-6412−0.15807 0.24032 0.02169 0.02769 0.30325 2.25994 0.02768 0.14334miR-1839-5p 0.18314 1.23480 0.20845 3.05761 0.30469 3.45981 0.165343.75248 miR-3102-3p 0.16636 1.08130 0.35127 4.01560 0.31541 2.933830.28527 4.06000 miR-344b-3p 0.06893 0.36837 0.16621 1.33117 0.438512.15959 0.26669 4.20000 miR-135a-5p −0.17353 0.30923 −0.00480 0.010790.56360 2.62229 0.30957 1.09472 miR-135a-2-3p 0.04867 0.22692 0.105530.51050 0.57250 2.50032 0.09612 0.81124 miR-7b-5p 0.12388 0.435450.05651 0.12626 0.64985 2.30313 0.15830 0.78228 *Only miRNAs with p <0.01 and log₂(FC) >±0.2 for either 2 or 4 months in Df(16)1/+ orDgcr8^(+/−) mutants are shown. FC, fold change in mutant vs. WT mice.Bold, miRNAs predicted to target Drd2 transcripts.The data discussed in this publication have been deposited in NCBI'sGene Expression Omnibus and are accessible through GEO Series accessionnumber GSE73981www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cpwxugcwdzsdhax&acc=GSE73981.

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The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference intheir entirety as if physically present in this specification.

LIST OF SEQUENCES SEQ ID NO: Type Source Sequence Description  1 RNAHomo sapiens UCCAGCAUCAGUGAUUUUGUUG mature miR-338-3p  2 DNA SyntheticTCCAGCATCAGTGATTTTGTTG forward primer for mmu- miR-338-3p analysis  3DNA Synthetic TTTTTCATTATTGCTCCTGACC forward primer for mmu-miR-335-3p analysis  4 DNA Synthetic TCAAGAGCAATAACGAAAAATGTforward primer for mmu- miR-335-5p analysis  5 DNA SyntheticTCAGCTCCTATATGATGCCTTT forward primer for mmu- miR-337-3p analysis  6DNA Synthetic CGGCGTCATGCAGGAGTTGATT forward primer for mmu-miR-337-5p analysis  7 DNA Synthetic TCAACAAAATCACTGATGCTGGforward primer for mmu- miR-3065-5p analysis  8 DNA SyntheticTCAGCACCAGGATATTGTTGGGGm forward primer for mmu- miR-3065-3p analysis  9DNA Synthetic GGATGTCATGATGTGCACAGC Drd2 forward primer formRNA analysis 10 DNA Synthetic CGCTTGCGGAGAACGATGDrd2 reverse primer for mRNA analysis 11 DNA SyntheticATGCTGGCCTGCCTGTGTTGT Aatk forward primer 12 DNA SyntheticAGGGGCAGGACATACACATCGG Aatk reverse primer 13 DNA SyntheticCGCTTCGGCAGCACATATAC U6 snRNA forward primer 14 DNA SyntheticTTCACGAATTTGCGTGTCAT U6 snRNA reverse primer 15 DNA SyntheticCTTTTGAACCCTTTTCCATCTG SnoRNA202 primer 16 DNA SyntheticTTAACAAAAATTCGTCACTACCA SnoRNA234 primer 17 DNA SyntheticGTACAGCTGTTGACAGTGAGCGACTCCAGCA miR-338-3p-1 primerTCAGTGATTTTGTTGTGTGAA 18 DNA Synthetic CCATCTGTGGCTTCACACAACAAAATCACTGmiR-338-3p-2 primer ATGCTGGAGTCGCTCACTGTCAACAGCT 19 DNA SyntheticGCCACAGATGGCAACAAAATCTGATGCTGGA miR-338-3p-3 primer GCTGCCTACTGCCTCGGAA20 DNA Synthetic AGCTTTCCGAGGCAGTAGGCAGCTCCAGCAT miR-338-3p-4 primerCAGATTTTGTTG 21 DNA Synthetic GTACAGCTGTTGACAGTGAGCGACTCAGCTCmiR-337-3p-1 primer CTATATGATGCCTTTTGTGAA 22 DNA SyntheticCCATCTGTGGCTTCACAAAAGGCATCATATA miR-337-3p-2 primerGGAGCTGAGTCGCTCACTGTCAACAGCT 23 DNA SyntheticGCCACAGATGGAAAGGCATCATAGGAGCTGA miR-337-3p-3 primer GCTGCCTACTGCCTCGGAA24 DNA Synthetic AGCTTTCCGAGGCAGTAGGCAGCTCAGCTCC miR-337-3p-4 primerTATGATGCCTTT 25 DNA Synthetic GTACAGCTGTTGACAGTGAGCGACCGGCGTCmiR-337-5p-1 primer ATGCAGGAGTTGATTTGTGAA 26 DNA SyntheticCCATCTGTGGCTTCACAAATCAACTCCTGCA miR-337-5p-2 primerTGACGCCGGTCGCTCACTGTCAACAGCT 27 DNA SyntheticGCCACAGATGGAATCAACTCGCATGACGCCG miR-337-5p-3 primer GCTGCCTACTGCCTCGGAA28 DNA Synthetic AGCTTTCCGAGGCAGTAGGCAGCCGGCGTCA miR-337-5p-4 primerTGCGAGTTGATT (SEQ ID NO: 28) 29 DNA SyntheticGTACAGCTGTTGACAGTGAGCGACTTTTTCA miR-335-3p-1 primerTTATTGCTCCTGACCTGTGAA 30 DNA Synthetic CCATCTGTGGCTTCACAGGTCAGGAGCAATAmiR-335-3p-2 primer ATGAAAAAGTCGCTCACTGTCAACAGCT 31 DNA SyntheticGCCACAGATGGGGTCAGGAGATAATGAAAAA miR-335-3p-3 primer GCTGCCTACTGCCTCGGAA32 DNA Synthetic AGCTTTCCGAGGCAGTAGGCAGCTTTTTCAT miR-335-3p-4 primerTATCTCCTGACC (SEQ ID NO: 32) 33 DNA SyntheticGTACAGCTGTTGACAGTGAGCGACTCAAGAG miR-335-5p-1 primerCAATAACGAAAAATGTTGTGAA 34 DNA Synthetic CCATCTGTGGCTTCACAACATTTTTCGTTATmiR-335-5p-2 primer TGCTCTTGAGTCGCTCACTGTCAACAGCT 35 DNA SyntheticGCCACAGATGGACATTTTTCGATTGCTCTTG miR-335-5p-3 primer AGCTGCCTACTGCCTCGGAA36 DNA Synthetic AGCTTTCCGAGGCAGTAGGCAGCTCAAGAGC miR-335-5p-4 primerAATCGAAAAATGT 37 DNA Synthetic CAACAAAATGCGGATGCTGGA miR-338-3p sponge38 DNA Synthetic GACACTGTGAGCGAAGACATA scrambled control 39 DNASynthetic ATAGCATACATTATACGAAGTTATCACTGG 5′ common reverse primer 40 DNASynthetic CTTCACTACACTCTCCCTAGTACAGTCTC 5′ gene-specific primer 41 DNASynthetic TCTAGAAAGTATAGGAACTTCCATGGTC 3′ common forward primer 42 DNASynthetic AGGAGACTCATAGTTCTCTGTATCATAGC 3′ gene-specific primer 43 DNAMus musculus ACGGCTGCCGGAGGGGCGGCCGTGCGTGGAT mouse Drd2 3′ UTRGCGGCGGGAGCTGGAAGCCTCGAGCAGCCGG (XM_006509996.2)CGCCTTCTCTGGCCCCGGGCGCCCTATGGCT TGAAGAGCCGTGCCACCCAGTGGCCCCACTGCCCCAATGGATCCACTGAACCTGTCCTGGTA CGATGATGATCTGGAGAGGCAGAACTGGAGCCGGCCCTTCAATGGGTCCGAAGGGAAGGCAG ACAGGCCCCACTACAACTACTATGCCATGCTGCTCACCCTCCTCATCTTTATCATCGTCTTT GGCAATGTGCTGGTGTGCATGGCTGTATCACGAGAGAAGGCTTTGCAGACCACCACCAACTA CCTGATAGTCAGCCTCGCTGTGGCCGATCTTCTGGTGGCCACACTGGTTATGCCCTGGGTCG TCTATCTGGAGGTGGTGGGTGAGTGGAAATTCAGCAGGATTCACTGTGACATCTTTGTCACT CTGGATGTCATGATGTGCACAGCAAGCATCTTGAACCTGTGTGCCATCAGCATCGACAGGTA CACAGCTGTGGCCATGCCTATGTTGTATAACACACGCTACAGCTCCAAGCGCCGAGTTACTG TCATGATCGCCATTGTCTGGGTCCTGTCCTTCACCATCTCTTGCCCACTGCTCTTTGGACTC AACAACACAGACCAGAATGAGTGTATCATTGCCAACCCTGCCTTCGTGGTCTACTCCTCCAT CGTCTCGTTCTACGTGCCCTTCATCGTCACCCTGCTGGTCTATATCAAAATCTACATCGTTC TCCGCAAGCGTCGGAAGCGGGTCAACACCAAGCGTAGCAGCCGAGCTTTCAGAGCCAACCTG AAGACACCACTCAAGGGCAACTGTACCCACCCTGAGGACATGAAACTCTGCACCGTTATCAT GAAGTCTAATGGGAGTTTCCCAGTGAACAGGCGGAGAATGGATGCTGCCCGCCGAGCTCAGG AGCTGGAAATGGAGATGCTGTCAAGCACCAGCCCCCCAGAGAGGACCCGGTATAGCCCCATC CCTCCCAGTCACCACCAGCTCACTCTCCCCGATCCATCCCACCACGGTCTACATAGCAACCC TGACAGTCCTGCCAAACCAGAAAAGAATGGGCATGCCAAGATTGTCAATCCCAGGATTGCCA AGTTCTTTGAGATCCAGACCATGCCCAATGGCAAAACCCGGACCTCCCTTAAGACGATGAGC CGCAGGAAGCTCTCCCAGCAGAAGGAGAAGAAAGCCACTCAGATGCTTGCCATTGTTCTTGG TGTGTTCATCATCTGCTGGCTGCCCTTCTTCATCACGCACATCCTGAATATACACTGTGACT GCAACATCCCACCAGTCCTCTACAGCGCCTTCACATGGCTGGGCTATGTCAACAGTGCCGTG AACCCCATCATCTATACCACCTTCAACATTGAGTTCCGCAAGGCCTTCATGAAGATCCTGCA CTGCTGAGTCTGCCCCTTGCCTGCACAGCAGCTGCTTGCCGCCTCCCTGCCTAGGCAGGCCA GACCTCATCCCTGCAAGCTGTGGGCAGAAAGGCCCAGATGGACTCGGCCTTCTCTTGACCCT GCAGGCTCTGCAGTGTTAGCTTGGCTCGGTGCCCCTCTCTGCCCACACACCCTTATCCTGCC AGGGTAGGGCCAGGGAGACTGGTATCTTACCAGCTCTGGGGTTGGATCCATGGCTCAGAGCA GCTCACAGAGTGCCCCTTTCACATGCAGATCCTGTCTCCTTGGCACCAAAGAAGCAGCAGCC TTCCTTGACCTTCCTCTCAGGCACGGAAGCTAGCTCAGTAGCGGAGCACACCTTGATTGTTG GCTTGGCCTGGCCCTTGCTTGCCTATGTTGGATCAGGTGGTAGAAGAGAAGGACAGTTCTTA CTTTACAGGGACCACATAGGAAAGCAGGGAACATGCCAAGGCCTCCAGGTGACGTTAGTGTC GGGAGACACACATAAACACCAGGTAGCTCCACGGACCCCAGAGAAACTGAGGCTGAAAATCT GTTTTCCACCCCAACTCTAGTGTGAATCCCTACTTTCCATAGCAGTGGGTATTGCTATGTTC TCCACTGTTATAGAATCCCATGGGGTTTCTGTACCTTCGGGGGAAAATAACTCTAATCCTCA AGGGCCCCAAGAGAGACTGTAAAGAGAAAAATAGCTGATTTCCCTCTACCCTCCAATCCACT CCGCCACTTCTTGACATACATTGGACATAGCCATTCCCCACAGCAGATGCTGGACAGCCTGG GAAGTTGAGCCTTGGACCAGTGTTGGAGCTGAAGTTGGAGGTGGTAACTTGGGGCTCTTGGG CGGGGGGTGTTGATATCTTCCCTCTTCCAAGTCTCTTCTCTGCCAGTGCCTCTGCCTTAGAG GAGGCTGTGGATGGGGCTGCTGGGGCTGCTGATACCATTGGGTCTGGCCCTGAGTGAGGGTG GGGAAGCTGCAGCTTGGAGGGGTCTGGGCTCCAACTCTGTAACATCACCATACATGCACCAA ACCAATAAAACCTTGACAAGAGTCATTCCCA CGG 44DNA Synthetic TCCAGCATCAGTGATTTTGTTG hsa-miR-338-3p primer

1-41. (canceled)
 42. A kit for determining the likelihood of developinga positive symptom of schizophrenia and/or for determining efficacy of atreatment for schizophrenia or 22q11 deletion syndrome comprising aprimer or a probe specific for miR-338-3p, and optionally furthercomprising instructions for use. 42-48. (canceled)
 49. A pharmaceuticalcomposition comprising miR-338-3p or a mimic or a functional derivativethereof and a pharmaceutically acceptable carrier or excipient. 50-51.(canceled)
 52. A pharmaceutical dosage form comprising miR-338-3p or amimic or a functional derivative thereof and a pharmaceuticallyacceptable carrier or excipient. 53-54. (canceled)
 55. The kit of claim42, further comprising miRNA isolation and/or purification means. 56.The kit of claim 42, wherein the primer or the probe comprises asequence that is at least 70% complementary to the sequenceUCCAGCAUCAGUGAUUUUGUUG (SEQ ID NO: 1).
 57. The pharmaceuticalcomposition of claim 49, wherein the functional derivative of miR-338-3pis a functional fragment of miR-338-3p or a variant thereof.
 58. Thepharmaceutical composition of claim 49, wherein the miR-338-3p or themimic or the functional derivative thereof is at least 7 nucleotides inlength.
 59. The pharmaceutical composition of claim 49, wherein themiR-338-3p or the mimic or the functional derivative thereof comprises asequence that has at least 70% sequence identity to the sequenceUCCAGCAUCAGUGAUUUUGUUG (SEQ ID NO: 1).
 60. The pharmaceuticalcomposition of claim 49, wherein the miR-338-3p or the mimic or thefunctional derivative thereof comprises one or more modificationsselected from modifications to the base moiety, modifications to thesugar moiety, modifications to the phosphate moiety, modifications tothe phosphate-sugar backbone, or any combination thereof.
 61. Thepharmaceutical composition of claim 49, wherein the miR-338-3p or themimic or the functional derivative thereof comprises a locked nucleicacid (LNA).
 62. The pharmaceutical composition of claim 49, wherein saidcomposition is suitable for an intranasal administration.
 63. Thepharmaceutical composition of claim 49, wherein said composition issuitable for a systemic administration.
 64. The pharmaceuticalcomposition of claim 49, wherein the composition is suitable foradministration to the thalamus.
 65. The pharmaceutical dosage form ofclaim 52, wherein the functional derivative of miR-338-3p is afunctional fragment of miR-338-3p or a variant thereof.
 66. Thepharmaceutical dosage form of claim 52, wherein the miR-338-3p or themimic or the functional derivative thereof is at least 7 nucleotides inlength.
 67. The pharmaceutical dosage form of claim 52, wherein themiR-338-3p or the mimic or the functional derivative thereof comprises asequence that has at least 70% sequence identity to the sequenceUCCAGCAUCAGUGAUUUUGUUG (SEQ ID NO: 1).
 68. The pharmaceutical dosageform of claim 52, wherein the miR-338-3p or the mimic or the functionalderivative thereof comprises one or more modifications selected frommodifications to the base moiety, modifications to the sugar moiety,modifications to the phosphate moiety, modifications to thephosphate-sugar backbone, or any combination thereof.
 69. Thepharmaceutical dosage form of claim 52, wherein the miR-338-3p or themimic or the functional derivative thereof comprises a locked nucleicacid (LNA).
 70. The pharmaceutical dosage form of claim 52, wherein thedosage form is a spray.